Env polypeptide complexes and methods of use

ABSTRACT

Provided herein are small molecule CD4 mimetics effective to bind to HIV Env proteins. A CD4 mimetic of the invention, when bound to an Env protein, is effective to induce a conformational change in the Env protein such that cyptic epitopes on the Env protein are exposed. Also provided herein are related methods of identifying and using such small molecule CD4 mimetics, for example, to elicit an immune response in a subject upon administration.

RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 11/597,337,filed Dec. 28, 2007, which is a U.S. national phase of PCT/US05/22808,filed Jun. 8, 2005, now WO05/121175, dated Dec. 22, 2005, which claimsthe benefit of U.S. Provisional Application No. 60/578,445, filed Jun.8, 2004, and U.S. Provisional Application No. 60/578,211, filed Jun. 8,2004. The above applications are incorporated herein in their entiretiesby reference.

STATEMENT OF GOVERNMENT SUPPORT

The application was made with support from the United States NIAID-NIHHIVRAD under Grant No. 5P01 AI48225-03. Thus, the U.S. Government mayhave certain rights in this invention.

TECHNICAL FIELD

The invention relates generally to CD4 mimetics including peptoids,small molecules, and peptoid-small molecule conjugates that bind to HIVEnv proteins and induce a conformational change in such Env proteins.More particularly, the invention relates to small molecules that induceconformational changes in Env polypeptides (such as monomeric oroligomeric gp120, gp140 or gp160) such that conserved, cryptic epitopesparticipating in Env-CD4 and chemokine receptor binding are exposed. Theinvention also pertains to methods of using these molecules to elicit animmune response against a broad range of HIV subtypes.

BACKGROUND

The human immunodeficiency virus (HIV-1, also referred to as HTLV-III,LAV or HTLV-III/LAV) is the etiological agent of the acquired immunedeficiency syndrome (AIDS) and related disorders. (see, e.g.,Barre-Sinoussi, et al., (1983) Science 220:868-871; Gallo et al. (1984)Science 224:500-503; Levy et al., (1984) Science 225:840-842; Siegal etal., (1981) N Engl. J. Med. 305:1439-1444; Guyader et al., (1987) Nature326:662-669).

The envelope protein of HIV-1, HIV-2 and SIV is a glycoprotein of about160 kd (gp160). During virus infection of the host cell, gp160 iscleaved by host cell proteases to form gp120 and the integral membraneprotein, gp41. The gp41 portion is anchored in the membrane bilayer ofvirion, while the gp120 segment protrudes into the surroundingenvironment. gp120 and gp41 are more covalently associated and freegp120 can be released from the surface of virions and infected cells.Furthermore, upon binding to its receptor, CD4, the Env polypeptideundergoes a significant structural rearrangement. After thisconformational change the CCR5 co-receptor binding site is exposed.Exposure of the CCR5 binding site, in turn, mediates viral entry intothe host cell. See, e.g., Wyatt, R., et al. (1998) Nature 393:705-711;Kwong, P., et al. (1998) Nature 393:648-659.

Env appears to be the primary target for inducing a humoral immuneresponse to HIV. However, it is known that antibodies directed againstgp120 do not generally exhibit broad antibody responses againstdifferent HIV strains and do not induce production of neutralizingantibodies. See, e.g., Javaherian, K., et al. (1989) Proc. Natl. Acad.Sci. 86:6786-6772; Matsushita, M., et al. (1988) J. Virol. 62:2107-2144;Putney, S., et al. (1986) Science 234:1392-1395; Rushe, J. R., et al.(1988) Proc. Nat. Acad. Sci. USA 85: 3198-3202; Matthews, T. (1986)Proc. Natl. Acad. Sci. USA. 83:9709-9713; Nara, P. L., et al. (1988) J.Virol. 62:2622-2628; Palker, T. J., et al. (1988) Proc. Natl. Acad. Sci.USA. 85:1932-1936).

Furthermore, although neutralizing antibodies are typically generated inthe course of HIV infection in humans, these antibodies do not providepermanent antiviral effect. This may in part be due to the generation of“neutralization escapes” virus mutants and to the general decline in thehost immune system associated with pathogenesis. See, e.g.,Barre-Sinoussi, F., et al. (1983) Science 220:868-871; Robert-Guroff,M., et al. (1985) Nature (London) 316:72-74; Weis, R., et al. (1985)Nature (London) 316:69-72; Weis, R., et al. (1986) Nature (London)324:572-575. Nonetheless, it is widely believed that the presence ofpre-existing neutralizing antibodies upon initial HIV-1 exposure willlikely have a protective effect, for instance by attaching to theincoming virions and reducing or preventing their infectivity for targetcells and prevent the cell-to-cell spread of virus in tissue culture.See, e.g., Hu et al. (1992) Science 255:456-459; Burton, D. R. andMontefiori, D. (1997) AIDS 11(suppl. A): 587-598; Montefiori and Evans(1999) AIDS Res. Hum. Ret. 15(8):689-698; Bolognesi, D. P., et al.(1994) Ann. Int. Med. 8:603-611; Haynes, B., F., et al. (1996) Science271:324-328.

Several categories of potentially effective neutralizing antibodies havebeen identified. For example, in most infected individuals, a subset ofbroadly reactive antibodies that interfere with binding of gp120 and CD4have been identified. See, e.g., Kang, C.-Y., et al. (1991) Proc. Natl.Acad. Set USA. 88:6171-6175; McDougal, J. S., et al. (1986) J. Immunol.137:2937-2944. Other antibodies are believed to bind to the chemokinereceptor-binding region after CD4 has bound to Env. See, e.g., Thali etal. (1993) J. Virol. 67:3978-3988). Monoclonal antibodies, such asIgG1b12, 2G12 (Mo et al. (1997) J. Virol 71:6869-6874), PA14 (Trkola etal. (2001) J. Virol. 75(2):579-88) and 2F5, which are directed to theCD4 binding site, also exhibit neutralizing effects. See, also, Trkolaet al. (1995) J. Virol. 69:6609-6617; D'Sousa et al (1997) J. Infect.Dis. 175:1062-1075. Furthermore, in order to generate antibodies againstthe CD4 binding site region, which is exposed only upon binding to CD4,several groups have attempted to generate neutralizing antibodies byadministering complexes of Env bound to CD4 (e.g., soluble CD4, referredto as “sCD4”) or to CD4 mimetics. See, e.g., Martin et al. (2003) Nat.Biotechnol. 21(1):71-6. However, administration of unadjuvanted sCD4alone is not immunogenic and adjuvanted sCD4 administration may triggerpotentially devastating autoimmune responses.

Therefore, there remains a need for additional molecules, which, whenadministered to a subject, are capable of triggering a conformationalchange in Env proteins such as gp120. This induced conformational changeis effective to expose cryptic epitopes on the Env protein to therebygenerate a neutralizing antibody response—without the negative effectsof adjuvanted sCD4.

SUMMARY

The present invention solves these and other problems by providing CD4mimetics that complex with Env to expose epitopes in or near the CD4binding site, and induce production of neutralizing antibodies. Alsoprovided are compositions and complexes of Env with the CD4 mimeticsdescribed herein, as well as antibodies directed against thesecomplexes.

In one aspect, the invention provides an immunogenic compositioncomprising an Env polypeptide and a small molecule CD4 mimetic having amolecular weight of less than about 1000 daltons. The CD4 mimeticpresent in the composition interacts with the Env polypeptide to form anEnv polypeptide/CD4 mimetic complex comprising an exposed crypticepitope.

Also provided is an immunogenic composition comprising an Envpolypeptide/CD4 mimetic complex arising from the Env polypeptide andsmall molecule CD4 mimetic described above.

In one embodiment of the invention, the binding affinity of a crypticepitope antibody such as Mab 17b or 48D to an Env polypeptide/CD4mimetic complex of the invention is greater than the binding affinity ofsuch cryptic epitope antibody to the Env polypeptide alone.

In another embodiment, an Env polypeptide/CD4 mimetic complex of theinvention exhibits at least 1% percent upregulation of the 17b epitopeor the 48D epitope when compared to the Env polypeptide alone.

Env polypeptides of the invention include polypeptides such as gp120,gp140 and gp160.

In another embodiment, the small molecule CD4 mimetic possesses amolecular weight selected from the following: about 900 daltons or less,about 800 daltons or less, about 750 daltons or less, about 700 daltonsor less, about 600 daltons or less, and about 500 daltons or less.

In a further embodiment, the small molecule is covalently attached,optionally via an intervening spacer, to a cross-linking moiety.

The invention further includes an immunogenic composition comprising asmall molecule that is crosslinked to an Env polypeptide to form acovalently bound Env polypeptide/CD4 mimetic complex.

In another embodiment, the small molecule CD4 mimetic comprises a fusedbicyclic or tricyclic core structure. Such core structures include anindole, a pyrrolopyridine, and a fluoren-9-one.

More particularly, in another embodiment, the small molecule comprises acore structure selected from:

where in structure V, none or one of ring carbons 4, 5, 6, or 7 may bereplaced by a nitrogen.

In an additional embodiment related to the above, in each of corestructures V and VI, one of the ring atoms is covalently attached, viaan optional linker, J, to a substituted or unsubstituted piperidine orpiperazine, which itself may form part of a bicyclic ring system such asa 1,2,3,4-tetrahydro-isoquinoline.

Additional embodiments related to the small molecule CD4 mimetics of theinvention include the following.

For example, in either of V and VI, one of the ring atoms may becovalently attached, via an optional linker, J, to3-oxomethylene-2-(1,2,3,4-tetrahydro-isoquinolin-1-ylmethyl)-2,3-dihydro-isoindol-1-one.

In a further embodiment, the core structure V is selected from the groupconsisting of 1-H indole, 1H-pyrrolo[2,3,-b]pyridine,1H-pyrrolo[2,3,-c]pyridine, 1H-pyrrolo[3,2,-b]pyridine, and1H-pyrrolo[3,2-c]pyridine.

In another embodiment, in each of core structures V and VI, any one ofthe ring atoms at position 2, 3, 6, or 7 is covalently attached, via anoptional linker, J, to a substituted or unsubstituted piperidine orpiperazine, which itself may form part of a bicyclic ring system.

In a further embodiment, in addition to the substituted or unsubstitutedpiperidine or piperazine, core structures V and VI each possess one ormore optional substituents at the remaining ring atom positions. Suchone or more optional substituents include C₁-C₆ alkyl, C₃-C₆ cycloalkyl,C₂-C₆ alkenyl, C₄-C₆ cycloalkenyl, C₂-C₆ alkynyl, halogen, —CN, phenyl,benzyl, nitro, C₁-C₆ alkoxy, C₃-C₆ cycloalkoxy, benzyloxy, —C(O)—C₁-C₆alkyl, —C(O)—C₆H₅—, and —C(O)—C₆H₄—W, wherein W is an ortho, meta, orpara-halogen.

In another embodiment, the optional linker, J, is from about one to 30atoms in length. Illustrative linkers include —C(O)CH₂— —C(O)—,—C(O)—CHCH₃—, —C(O)—C(O)—, —CH(OH)CH₂—, —CH₂—, —N—C(O)-furanyl, ineither orientation.

In another embodiment, a small molecule CD4 mimetic in accordance withthe invention includes compounds I-30 herein.

In another embodiment, a composition of the invention further includesan adjuvant.

In another aspect, the invention includes a method of eliciting animmune response in a subject. In the method, any one or more of theherein described immunogenic compositions is administered to a mammaliansubject to thereby elicit an immune response therein, wherein the Envprotein and CD4 mimetic are administered simultaneously, sequentially inany order, separately, or as a complex.

In a particular embodiment, the method includes administering an Envpolypeptide such as a gp120 polypeptide.

In another embodiment, the method includes administering an Envpolypeptide and a small molecule CD4 mimetic such that the CD4 mimeticinteracts with the Env polypeptide to expose the CD41 cryptic epitope onthe resulting Env polypeptide/CD4 mimetic complex. In a relatedembodiment, the method is effective to elicit in a mammalian subject thecellular production of antibodies to CD41.

In another aspect, also provided is a method of identifying a CD4mimetic capable of interacting with an Env polypeptide to expose acryptic epitope on the Env polypeptide. The method includes the steps ofa) providing an Env polypeptide; b) providing a candidate small moleculehaving a molecular weight of less than about 1000 daltons; c) contactingthe Env polypeptide with the candidate molecule to result in an Envpolypeptide-candidate small molecule mixture; d) measuring the affinityof a cryptic epitope antibody to bind to the Env polypeptide-candidatesmall molecule mixture formed in step c); and e) based upon the affinitymeasured in step d), selecting a candidate molecule effective to form anEnv polypeptide-candidate small molecule complex whose binding affinityto a cryptic epitope antibody is greater than or equal to a knowncryptic epitope antibody antigen.

In one embodiment of the above aspect of the invention, the screeningmethod further includes the step of crosslinking the candidate smallmolecule to the Env polypeptide to provide a covalently linked Envpolypeptide/candidate small molecule complex.

In another embodiment of this aspect, the method further includes thestep of comparing the binding affinity of the cryptic epitope antibodyand the Env polypeptide/candidate small molecule complex to the bindingaffinity of the cryptic epitope antibody and the Env polypeptide per se,or to the binding affinity of the cryptic epitope antibody and an Envpolypeptide/CD4 polypeptide complex.

Various embodiments of the screening method include those in which theEnv polypeptide is a gp120 polypeptide, and/or where the candidatemolecule is, for example, a known competitive CD4 inhibitor of a gp120polypeptide, a known β turn mimetic, or is a member of a small moleculelibrary, wherein the members of the library comprise a fused bicyclic ortricyclic core structure, among others.

In another embodiment of the screening method, the cryptic epitopeantibody is bound to a solid support, e.g., one that comprises a goldfilm surface.

In another aspect, the invention is directed to a method of producing anantibody that binds to a cryptic epitope of an Env polypeptide. In sucha method, an immunological composition of the invention is administeredto a mammalian subject under conditions effective to form antibodies inthe subject.

A further embodiment of the above includes a method that includes thestep of isolating the antibodies formed by the subject (for example,neutralizing antibodies, or monoclonal antibodies, or polyclonalantibodies).

Also provided is the use of the Env polypetide and a small molecule CD4mimetic having any one or more of the herein described features in thepreparation of a medicament for treating or preventing an Envpolypeptide-mediated disease.

The following numbered embodiments are encompassed by the presentinvention:

1. An immunogenic composition comprising an Env polypeptide and a CD4mimetic, wherein said CD4 mimetic interacts with said Env polypeptide toform an Env polypeptide/CD4 mimetic complex to expose cryptic epitopeson the Env polypeptide/CD4 mimetic complex.2. The immunogenic composition of Embodiment 1, wherein the bindingaffinity of a cryptic epitope antibody to said Env polypeptide/CD4mimetic complex is greater than the binding affinity of said crypticepitope antibody to said Env polypeptide alone.3. The immunogenic composition of Embodiment 2, wherein said crypticepitope antibody is MAbs 17b or 48D.4. The immunogenic composition of Embodiment 1, wherein said Envpolypeptide/CD4 mimetic complex exhibits at least 1% percentupregulation of the 17b epitope compared to the Env polypeptide alone.5. The immunogenic composition of Embodiment 1, wherein said Envpolypeptide is a gp120 polypeptide.6. The immunogenic composition of Embodiment 1, wherein said CD4 mimeticis a peptoid.7. The immunogenic composition of Embodiment 6, wherein said peptoid hasa molecular weight of less than about 2000 daltons.8. The immunogenic composition of Embodiment 7, wherein said peptoid isfurther conjugated to a small molecule to form a peptoid-small moleculeconjugate.9. The immunogenic composition of Embodiment 8, wherein said peptoidsmall molecule conjugate is crosslinked to said Env polypeptide, to forma covalently crosslinked Env polypeptide/CD4 mimetic complex.10. The immunogenic composition of Embodiment 1, wherein said CD4mimetic is a small molecule having a molecular weight of less than about1000 daltons.11. The immunogenic composition of Embodiment 10, wherein said smallmolecule is crosslinked to said Env polypeptide, to form a covalentlybound Env polypeptide/CD4 mimetic complex.12. The immunogenic composition of Embodiment 10, wherein said smallmolecule is a competitive CD4 inhibitor of a gp120 polypeptide.13. The immunogenic composition of Embodiment 1, wherein said CD4mimetic corresponds to one of Structures I, II, or III, includingstereoisomers and salts thereof.

where in each of structures I, II and III:

W, X, Y, and Z are each independently selected from the group consistingof carbon and nitrogen, provided that no more than one of W, X, Y, or Zis nitrogen;

R₁, R′₁, R₂, R₃, R₄, R₅ are each independently selected from the groupconsisting of H, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, C₂-C₆ alkenyl, C₄-C₆cycloalkenyl, C₂-C₆ alkynyl, halogen, CN, phenyl, nitro, OC(O)R₁₅,C(O)R₁₅, C(O)OR₁₆, C(O)NR₁₇R₁₈, OR₁₉, SR₂₀, and NR₂₁R₂₂, each of saidalkyl and cycloalkyl being optionally substituted with one to threesubstituents independently selected from halogen, CN, NO₂, OC(O)R₁₅,C(O)R₁₅, C(O)OR₁₅, C(O)NR₁₇R₁₈, OR₁₉, SR₂₀, and NR₂₁R₂₂, with theproviso that R₂, R₃, R₄, or R₅ is absent when the ring atom to which itis attached is a double-bonded nitrogen, i.e., a trivalent nitrogen;

R₁₅, is independently selected from the group consisting of H, C₁-C₆alkyl, C₃-C₆ cycloalkyl, C₂-C₆ alkenyl, and C₄-C₆ cycloalkenyl;

R₁₆, R₁₉, and R₂₀ are each independently selected from the groupconsisting of H, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, C₂-C₆ alkenyl, C₄-C₆cycloalkenyl, C₃-C₆ alkynyl, phenyl, and benzyl, wherein each of saidalkyl, cycloalkyl, phenyl, and benzyl are optionally substituted withone, two, or three substituents selected from the group consisting ofhalogen, NR₂₁R₂₂, OH, CN, or NO₂; provided the carbon atoms thatcomprise the carbon-carbon triple bond of said C₃-C₆ alkynyl are not thepoint of attachment to the oxygen or sulfur to which R₁₆, R₁₉, or R₂₀ isattached;

R₁₇ and R₁₈ are each independently selected from the group consisting ofH, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, C₃-C₆ alkenyl, C₄-C₆ cycloalkenyl, andC₃-C₆ alkynyl, wherein each of said alkyl and cycloalkyl are optionallysubstituted with one, two, or three substituents selected from the groupconsisting of halogen, NR₂₁R₂₂, OH, CN, or NO₂; provided the carbonatoms that comprise the carbon-carbon double bond of said C₃-C₆ alkenylor the carbon-carbon triple bond of said C₃-C₆ alkynyl are not the pointof attachment to the nitrogen to which R₁₇ and R₁₈ is attached;

R₂₁ and R₂₂ are each independently selected from the group consisting ofH, OH, C₆ alkyl, C₃-C₆ cycloalkyl, C₃-C₆ alkenyl, C₅-C₆ cycloalkenyl,C₃-C₆ alkynyl, phenyl, and C(O)R₂₃; provided the carbon atoms thatcomprise the carbon-carbon double bond of said C₃-C₆ alkenyl, C₄-C₆cycloalkenyl, or the carbon-carbon triple bond of said C₃-C₆ alkynyl arenot the point of attachment to the nitrogen to which R₂₁ and R₂₂ isattached;

R₂₃ is selected from the group consisting of H, C₁-C₆ alkyl, C₃-C₆cycloalkyl, C₂-C₆ alkenyl, C₄-C₆ cycloalkenyl, and C₂-C₆ alkynyl;

R₆ is (CH₂)_(n)Q, wherein n is 0-6;

Q is selected from:

-   -   (1) H, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, C₂-C₆ alkenyl, C₄-C₆        cycloalkenyl, C₃-C₆ alkynyl, halogen, CN, Ar, NO₂, C(O)R₂₄,        C(O)OR₂₅, C(O)NR₂₆R₂₇, COOAr, TR₆, NR₂₁R₂₂, NC(O)NR₂₁R₂₂,        OC(O)R₁₆, (N(R₂₁)₂)C═N-T-R₂₂, C(O)Ar, or S(O)_(m)R₂₁;    -   (2) a 4-7 membered heterocyclic ring, optionally substituted        with R₁₆, which may contain 1-3 heteroatoms selected from the        group consisting of O, S, SO, SO₂, N, and NR₂₄;

T is S or O;

m is 0-2;

R₂₄ is selected from the group consisting of H, C₁-C₆ alkyl, C₃-C₆cycloalkyl, C₃-C₆ alkenyl, C₄-C₆ cycloalkenyl, and C₃-C₆ alkynyl;

R₂₅ is selected from the group consisting of C₁-C₆ alkyl, C₃-C₆cycloalkyl, C₂-C₆ alkenyl, C₄-C₆ cycloalkenyl, and C₃-C₆ alkynyl;provided the carbon atoms that comprise the carbon-carbon triple bond ofsaid C₃-C₆ alkynyl are not the point of attachment to the oxygen towhich R₂₅ is attached;

R₂₆ and R₂₇ are each independently selected from the group consisting ofH, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, C₃-C₆ alkenyl, C₅-C₆ cycloalkenyl, andC₃-C₆ alkynyl; provided the carbon atoms that comprise the carbon-carbondouble bond of said C₃-C₆ alkenyl, C₅-C₆ cycloalkenyl, or thecarbon-carbon triple bond of said C₃-C₆ alkynyl are not the point ofattachment to the nitrogen to which R₂₆ and R₂₇ are attached;

R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃, and R₁₄ are each independently selectedfrom the group consisting of H, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, C₂-C₆alkenyl, C₄-C₆ cycloalkenyl, C₂-C₆ alkynyl, CR₂₈R₂₉OR₃₀, C(O)R₃₁,CR₃₂(OR₃₃)OR₃₄, CR₃₅NR₃₆R₃₇, C(O)OR₃₈, C(O)NR₃₉R₄₀, CR₄₁R₄₂F, CR₄₃F₂,and CF₃, each of said alkyl and cycloalkyl being optionally substitutedwith one to three substituents independently selected from halogen, CN,NO₂, OC(O)R₁₅, C(O)R₁₅, C(O)OR₁₅, C(O)NR₁₇R₁₈, OR₁₉, SR₂₀, and NR₂₁R₂₂,or R₈ and R₉ may be absent such that the carbon ring atoms to which eachis independently attached together form a double bond.

R₂₈, R₂₉, R₃₀, R₃₁, R₃₂, R₃₅, R₄₁, R₄₂ and R₄₃ are each independentlyselected from the group consisting of H, C₁-C₆ alkyl, C₃-C₆ cycloalkyl,C₂-C₆ alkenyl, C₄-C₆ cycloalkenyl, C₂-C₆ alkynyl, and C(O)R₄₄;

R₃₃, R₃₄ and R₃₈ are each independently selected from the groupconsisting of H, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, C₃-C₆ alkenyl, C₄-C₆cycloalkenyl, and C₃-C₆ alkynyl; provided the carbon atoms that comprisethe carbon-carbon triple bond of said C₃-C₆ alkynyl are not the point ofattachment to the oxygen to which R₃₄ and R₃₈ are attached;

R₃₆ and R₃₇ are each independently selected from the group consisting ofH, C₁-C₆ alkyl, C₁-C₆ cycloalkyl, C₃-C₆ alkenyl, C₄-C₆ cycloalkenyl, andC₃-C₆ alkynyl; provided the carbon atoms that comprise the carbon-carbontriple bond of said C₃-C₆ alkynyl are not the point of attachment to thenitrogen to which R₃₆ and R₃₇ are attached;

R₃₉ and R₄₀ are each independently selected from the group consisting ofH, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, C₂-C₆ alkenyl, C₄-C₆ cycloalkenyl, andC₃-C₆ alkynyl; provided the carbon atoms that comprise the carbon-carbontriple bond of said C₃-C₆ alkynyl are not the point of attachment to thenitrogen to which R₃₉ and R₄₀ are attached; R₄₄ is selected from thegroup consisting of H, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, C₂-C₆ alkenyl,C₄-C₆ cycloalkenyl, and C₂-C₆ alkynyl;

J is —(CO)—, —(CO)CH₂—, —(CO)(CO)—, or —(CO)CHR₉—;

U is nitrogen or CH;

L is —(CO)— or —CH₂—;

R_(A), R_(B), and R_(C) are independently selected from the groupconsisting of Ar, H, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, C₂-C₆ alkenyl, C₄-C₆cycloalkenyl, C₂-C₆ alkynyl, halogen, CN, nitro, OC(O)R₁₅, C(O)R₁₅,C(O)OR₁₆, C(O)NR₁₇R₁₈, OR₁₉, SR₂₀ and NR₂₁R₂₂, each of said alkyl andcycloalkyl being optionally substituted with one to three substituentsindependently selected from halogen, CN, NO₂, OC(O)R₁₅, C(O)R₁₅,C(O)OR₁₅, C(O)NR₁₇R₁₈, OR₁₉, SR₂₀, and NR₂₁R₂₂;

Ar is a 5-12 membered aromatic ring optionally containing one to fiveheteroatoms independently selected from the group consisting of O, S, N,or NR₁₆ and optionally substituted with one to six substitutentsindependently selected from the group consisting of R₄₅, R₄₆, R₄₇, R₄₈,or R₄₉;

R₄₅ R₄₆, R₄₇, R₄₈, and R₄₉ are each independently H, C₁-C₆ alkyl, C₃-C₆cycloalkyl, C₂-C₆ alkenyl, C₃-C₆ cycloalkenyl, C₂-C₆ alkynyl, halogen,CN, nitro, C(O)R₄₅, COOR₄₆, hydroxyl, hydroxymethyl, trifluoromethyl,trifluoromethoxy, O-benzyl, O-phenyl, OC(O)C₁-C₆ alkyl, SC(O)C₁-C₆alkyl, S(O)_(m)C₁-C₆ alkyl, S(O)₂NR₂₁R₂₂, O—V, CH₂ (CH₂)_(p)—V,O(CH₂)—V, (CH₂)_(p)O—V, CH═CH—V, or TR₄₇, each of said alkyl andcycloalkyl being optionally substituted with one to three substitutentsindependently selected from the group consisting of, NR₂₁NR₂₂, OH, CN,or NO₂;

V is 4-methoxyphenyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, pyrazyl,quinolyl, 3,5-dimethylisoxazoyl, isoxazoyl, 2-methylthiazoyl, thiazoyl,2-thienyl, 3-thienyl, or pyrimidyl;

p is 1-2;

R₄₅ and R₄₆ are each independently H, C₁-C₆ alkyl, or C₃-C₆ cycloalkyl;

R₄₇ is H, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, C₂-C₆ alkenyl, C₃-C₆cycloalkenyl, C₂-C₆ alkynyl, C(O)R₄₈ or C(O)OR₄₉, each of said alkyl andcycloalkyl being optionally substituted with one to three substitutentsindependently selected from the group consisting of, NR₂₁NR₂₂, OH, CN,or NO₂; and

R₄₈, R₄₉ are each independently H, C₁-C₆ alkyl, or C₃-C₆ cycloalkyl,each of said alkyl and cycloalkyl being optionally substituted with oneto three substitutents independently selected from the group consistingof, NR₂₁NR₂₂, OH, CN, or NO₂,

provided that said compound is not1-[4-(methyloxy)-1H-pyrrolo[2,3-b]pyridin-3-yl]-2-[(2R)-2-methyl-4-(phenylcarbonyl)piperazin-1-yl]-2-oxoethanone,1-[4,7-bis(methyloxy)-1H-indol-3-yl]-2-oxo-2-[4-(phenylcarbonyl)piperazin-1-yl]ethanone,or 1-(1H-indol-3-yl)-2-oxo-2-[4-(phenylcarbonyl)piperazin-1-yl]ethanone.

14. The immunogenic composition of Embodiment 13, wherein J is —(CO)CH₂—or —(CO)CHR₉—.15. The immunogenic composition of Embodiment 13, wherein J is —(CO)—.16. The immunogenic composition of Embodiment 13, wherein U is nitrogenand L is —CH₂—.17. The immunogenic composition of Embodiment 13, wherein U is nitrogenand L is —(CO)—.18. The immunogenic composition of Embodiment 13, wherein W, X, Y, and Zare carbon.19. The immunogenic composition of Embodiment 13, wherein n is 0 and Qis H or C₁-C₆ alkyl.20. The immunogenic composition of Embodiment 13, wherein R_(A), R_(B),and R_(C) are independently selected from the group consisting of Ar,C(O)OR₁₆, and NR₂₁R₂₂.21. The immunogenic composition of Embodiment 13, wherein Ar is selectedfrom the group consisting of

wherein:

A₁, A₂, A₃, A₄, A₅, B₁, B₂, B₃, B₄, C₁, C₂, C₃, D₁, D₂, and D₃ are eachindependently selected from the group consisting of H, CN, halogen, NO₂,C₁-C₆ alkyl, C₃-C₆ cycloalkyl, C₂-C₆ alkenyl, C₄-C₆ cycloalkenyl, C₂-C₆alkynyl, OR₅₀, NR₅₁R₅₂, SR₅₃, N₃, and CH(—N═N—)CF₃;

R₅₀ is selected from the group consisting of H, C₁-C₆ alkyl, C₃-C₆cycloalkyl, C₂-C₆ alkenyl, C₄-C₆ cycloalkenyl, and C₃-C₆ alkynyl;provided the carbon atoms that comprise the carbon-carbon triple bond ofsaid C₃-C₆ alkynyl are not the point of attachment to the oxygen towhich R₅₀ is attached;

R₅₁ and R₅₂ are each independently selected from the group consisting ofH, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, C₃-C₆ alkenyl, C₅-C₆ cycloalkenyl,C₃-C₆ alkynyl, and C(O)R₅₅; provided the carbon atoms that comprise thecarbon-carbon double bond of said C₅-C₆ alkenyl, C₄-C₆ cycloalkenyl, orthe carbon-carbon triple bond of said C₃-C₆ alkynyl are not the point ofattachment to the nitrogen to which R₅₁ and R₅₂ are attached;

R₅₃ is selected from the group consisting of H, C₁-C₆ alkyl, C₃-C₆cycloalkyl, C₂-C₆ alkenyl, C₄-C₆ cycloalkenyl, C₃-C₆ alkynyl andC(O)R₅₄; provided the carbon atoms that comprise the carbon-carbontriple bond of said C₃-C₆ alkynyl are not the point of attachment to thesulfur to which R₅₃ is attached;

R₅₄ is C₁-C₆ alkyl or C₃-C₆ cycloalkyl; and

R₅₅ is selected from the group consisting of H, C₁-C₆ alkyl, and C₃-C₆cycloalkyl.

22. The immunogenic composition of Embodiment 13, wherein said CD4mimetic is selected from the group consisting of1-[2-(5-methoxy-1H-indol-3-yl)-2-oxoethyl]piperidine-4-carboxamide,2-(4-benzylpiperazin-1-yl)-1-(5-methoxy-1H-indol-3-yl)ethanone,{1-[(4,6-dimethyl-1H-indol-2-yl)carbonyl]piperidin-4-yl}(4-fluorophenyl)methanone,1-[2-(5-methoxy-1,2-dimethyl-1H-indol-3-yl)-1-methyl-2-oxoethyl]piperidine-4-carboxamide,ethyl-1-[(4,6-dimethyl-1H-indol-2-yl)carbonyl]piperidine-4-carboxylate,6-{[4-(2,6-difluorobenzoyl)piperazin-1-yl]carbonyl}-1H-indole,2-{[4-(1H-indol-6-ylcarbonyl)piperazin-1-yl]carbonyl}phenyl acetate,6-{[4-(3,4,5-trimethoxybenzoyl)piperazin-1-yl]carbonyl}-1H-indole,1-[2-(5-methoxy-1,2-dimethyl-1H-indol-3-yl)-2-oxoethyl]piperidine-4-carboxamide,and 4-{[4-(2-furoyl)piperazin-1-yl]carbonyl}-9H-fluoren-9-one.23. The immunogenic composition of Embodiment 1, further comprising anadjuvant.24. A method of eliciting an immune response in a subject, the methodcomprising administering an Env polypetide and a CD4 mimetic to saidsubject, wherein said CD4 mimetic interacts with said Env polypetide toexpose cryptic epitopes on an Env polypeptide/CD4 mimetic complex.25. The method of Embodiment 24, wherein said Env polypetide and saidCD4 mimetic are administered intramuscularly or subcutaneously.26. The method of Embodiment 24, wherein the exposed cryptic epitope isCD41.27. The method of Embodiment 24, wherein said immune response is thecellular production of antibodies to CD41.28. The method of Embodiment 24, wherein said Env polypeptide is a gp120polypeptide.29. The method of Embodiment 24, wherein said CD4 mimetic is a CD4mimetic according to any one of embodiments 5-21.30. A method of identifying a CD4 mimetic capable of interacting with anEnv polypeptide to expose cryptic epitopes on said Env polypeptide, themethod comprising the steps of:

-   -   a) providing an Env polypeptide;    -   b) providing a compound;    -   c) contacting said Env polypeptide with said compound;    -   d) measuring the affinity of a cryptic epitope antibody to bind        to an Env polypeptide/compound complex; and    -   e) selecting said compound.        31. The method of Embodiment 30, further comprising the step of        crosslinking said compound to said Env polypeptide to provide a        covalently linked Env polypeptide/compound complex.        32. The method of Embodiment 30, further comprising the step of        comparing the binding affinity of said cryptic epitope antibody        and said Env polypeptide/compound complex to    -   a) the binding affinity of said cryptic epitope antibody and the        Env polypeptide, or to    -   b) the binding affinity of said cryptic epitope antibody and an        Env polypeptide/CD4 polypeptide complex.        33. The method of Embodiment 30, wherein said Env polypeptide is        a gp120 polypeptide.        34. The method of Embodiment 30, wherein said compound is a        known competitive CD4 inhibitor of a gp120 polypeptide.        35. The method of Embodiment 30, wherein said compound is a        known 13 turn mimetic.        36. The method of Embodiment 30, wherein said compound is a        member of a small molecule or peptoid library.        37. The method of Embodiment 30, wherein said cryptic epitope        antibody is selected from the group consisting of MAbs 17b and        48D.        38. The method of Embodiment 30, wherein said cryptic epitope        antibody is bound to a solid support.        39. The method of Embodiment 38, wherein the surface of said        solid support comprises a gold film.        40. A method of producing antibodies that bind to cryptic        epitopes of an Env polypeptide, the methods comprising the step        of:    -   a) administering an immunological composition according to any        of Embodiments 1-22 to a subject under conditions that allow        production of antibodies in the subject.        41. The method of Embodiment 40, further comprising the step of        isolating the antibodies produced in the subject.        42. The method of Embodiments 40 or 41, wherein the antibodies        are neutralizing antibodies.        43. The method of Embodiments 40, 41, and 42, wherein the        antibodies are monoclonal antibodies.        44. The method of Embodiments 40, 41, and 42, wherein the        antibodies are polyclonal antibodies.        45. Use of the Env polypetide and the CD4 mimetic of any one of        Embodiments 5-22 in the preparation of a medicament for treating        or preventing an Env polypetide mediated disease.

In another aspect, the invention includes complexes of any HIV Envpolypeptide and any of the small molecule CD4 mimetics described herein.In certain embodiments, the HIV Env polypeptide is based on strainSF162. In certain embodiments, the HIV Env polypeptide comprises one ormore amino acid deletions in V1, V2, V3, V4 and/or V5. In yet anotherembodiment, the polypeptide comprises an HIV Env polypeptide (e.g.,native or modified gp160, gp140, oligomeric-gp140, gp120) complexed to aCD4 mimetic. The HIV Env polypeptide and CD4 protein can be complexed bycrosslinking (e.g., using formaldehyde); using a fixative (e.g.,formalin); and/or can complex spontaneously under suitable conditions.

In another aspect, the invention includes a method of producingantibodies that bind to cryptic epitopes of HIV Env, the methodscomprising the step of administering any of the CD4 mimetics describedherein to a subject under conditions that allow production of antibodies(e.g., neutralizing antibodies, monoclonal antibodies, polyclonalantibodies) in the subject. In certain embodiments, the antibodiesproduced by the subject are then isolated.

In another aspect, the invention includes immunogenic compositionscomprising any of the mimetics, complexes and/or antibodies describedherein. In certain embodiments, the immunogenic compositions furthercomprise one or more adjuvants.

In yet another aspect, the invention includes a method of inducing animmune response (e.g., a humoral response such as a neutralizingantibody response and/or a cellular immune response) in subjectcomprising, administering any of the mimetics and/or complexes describedherein to a subject in an amount sufficient to induce an immune responsein the subject.

In certain embodiments, the method comprises (a) administering a firstcomposition comprising any of the mimetics, complexes and/or antibodiesdescribed herein in a priming step and (b) administering a secondcomposition comprising any of the mimetics, complexes and/or antibodiesdescribed herein, as a booster, in an amount sufficient to induce animmune response in the subject.

In any of the methods described herein, the subject can be a mammal, forexample a human or non-human mammal and the introduction can beintramuscularly, intramucosally, intranasally, subcutaneously,intradermally, transdermally, intravaginally, intrarectally, orallyand/or intravenously.

These and other embodiments of the subject invention will readily occurto those of skill in the art in light of the disclosure herein.

Each and every feature of the invention described herein is meant toapply equally to each and every embodiment of the invention even if notexplicitly stated.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting inhibition of CD4 binding to Env uponbinding of a representative CD4 mimetic of the invention, ethyl1-[(4,6-dimethyl-1H-indol-2-yl)carbonyl]piperidine-4-carboxylate, 5, asdetermined by surface plasmon resonance (SPR) analysis (Example 1);

FIG. 2 is a graph depicting upregulation in binding of monoclonalantibody 17b (MAb 17b) to an illustrative CD4 mimetic-Env complex of theinvention in comparison to binding to a sCD4-gp120SF162 complex(Examples 1 and 4). Monoclonal 17b recognizes an epitope that is exposedupon binding of Env to CD4;

FIG. 3 is a table containing a compilation of screening data forrepresentative small molecule CD4 mimetics in accordance with theinvention as described in detail in Example 6;

FIGS. 4A and 4B are graphs demonstrating the inhibition of CD4 bindingto gp120 and the upregulation of CD4 inducible epititopes on gp120,respectively, for an illustrative small molecule CD4 mimetic of theinvention, 26,2-[4-(2-methoxy-phenyl)-piperazin-1-yl]-1-(2-methyl-1H-indol-3-yl)-ethanone(Example 6);

FIGS. 5A and 5B are graphs illustrating the inhibition of CD4 binding togp120, and the absence of upregulation of CD4 inducible epititopes ongp120, respectively, for an illustrative small molecule CD4 mimetic ofthe invention, 12,[4-(1H-indol-3-yl)-piperidin-1-yl]-(3,4,5-trimethoxy-phenyl)-methanone(Example 6); and

FIGS. 6A, 6B, and 6C are graphs illustrating the non-inhibition of CD4binding to gp120 (FIG. 6A), and the upregulation of CD4 inducibleepitopes on gp120 as indicated by extent of binding to the monoclonalantibodies 17b (FIG. 6B) and 48D (FIG. 6C), respectively, for anillustrative small molecule CD4 mimetic of the invention,2,2-(4-benzylpiperazin-1-yl)-1-(5-methoxy-1H-indol-3-yl)ethanone.

DETAILED DESCRIPTION

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, viral immunobiology,molecular biology and recombinant DNA techniques within the skill of theart. Such techniques are explained fully in the literature. See, e.g.,T. E. Creighton, Proteins: Structures and Molecular Properties (W.H.Freeman and Company, 1993); Nelson L. M. and Jerome H. K. HIV Protocolsin Methods in Molecular Medicine, vol. 17, 1999; Sambrook, et al.,Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory,1989); F. M. Ausubel et al. Current Protocols in Molecular Biology,Greene Publishing Associates & Wiley Interscience New York; andLipkowitz and Boyd, Reviews in Computational Chemistry, volumes1-present (Wiley-VCH, New York, N.Y., 1999).

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a”, “an” and “the” include plural referentsunless the content clearly dictates otherwise. Thus, for example,reference to “a CD4 mimetic” includes a mixture of two or more mimetics,and the like.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

DEFINITIONS

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

The term “CD4 mimetic” refers to a molecule that interacts with an Envpolypeptide (e.g., gp120, gp140, gp160), preferably such that functionalepitopes (e.g., cryptic epitopes) in or near the CD4 and/or chemokinereceptor binding sites(s) are exposed. Cryptic epitopes are alsoreferred to herein as inducible epitopes.

The term “small molecule” refers to an organic compound eithersynthesized in the laboratory or found in nature. Typically, a smallmolecule is characterized in that it contains several carbon-carbonbonds, and has a molecular weight of less than 2500, preferably lessthan 2000, even more preferably less than about 1500, still morepreferably less than about 1500, even more preferably less than about1000, and most preferably less than about 750. A small molecule isnon-macromolecular in nature, that is to say, does not contain amultiple repetitions (e.g., more than 5 repeat units) of a particulartype of monomer subunit, such as in the case of macromolecules such asproteins, and other polymers. Examples of “small molecules” that occurin nature include, but are not limited to, taxol, dynemicin, andrapamycin. Examples of “small molecules” that are synthesized in thelaboratory include, but are not limited to, the CD4 mimetics describedherein. Small molecules may be readily prepared by synthetic organictechniques, such as by combinatorial chemistry techniques. See, e.g.,U.S. Pat. No. 6,448,443. A small molecule CD4 mimetic in accordance withthe invention excludes the following:1-[4-(methyloxy)-1H-pyrrolo[2,3-b]pyridin-3-yl]-2-[(2R)-2-methyl-4-(phenylcarbonyl)piperazin-1-yl]-2-oxoethanone,1-[4,7-bis(methyloxy)-1H-indol-3-yl]-2-oxo-2-[4-(phenylcarbonyl)piperazin-1-yl]ethanone,and1-(1H-indol-3-yl)-2-oxo-2-[4-(phenylcarbonyl)piperazin-1-yl]ethanone.

The term “acylamino” as used herein refers to an acyl (CO—) radical towhich an amino group is appended.

The term “loweralkyl” as used herein refers to branched or straightchain acyclical alkyl groups comprising one to ten carbon atoms,including, e.g., methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl,neopentyl and the like.

The phrase “alkyl” refers to alkyl groups that do not containheteroatoms. Thus the phrase includes straight chain alkyl groups suchas methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl,decyl, undecyl, dodecyl and the like. The phrase also includes branchedchain isomers of straight chain alkyl groups, including but not limitedto, the following that are provided by way of example: —CH(CH₃)₂,—CH(CH₃)(CH₂CH₃), —CH(CH₂CH₃)₂, — C(CH₃)₃, —C(CH₂CH₃)₃, —CH₂CH(CH₃)₂,—CH₂CH(CH₃)(CH₂CH₃), —CH₂CH(CH₂CH₃)₂, —CH₂C(CH₃)₃, —CH₂C(CH₂CH₃)₃,—CH(CH₃)CH(CH₃)(CH₂CH₃), —CH₂CH₂CH(CH₃)₂, —CH₂CH₂CH(CH₃)(CH₂CH₃),—CH₂CH₂CH(CH₂CH₃)₂, —CH₂CH₂C(CH₃)₃, —CH₂CH₂C(CH₂CH₃)₃,—CH(CH₃)CH₂CH(CH₃)₂, —CH(CH₃)CH(CH₃)CH(CH₃)₂,—CH(CH₂CH₃)CH(CH₃)CH(CH₃)(CH₂CH₃), and others. Preferred unsubstitutedalkyl groups include straight and branched chain alkyl groups and cyclicalkyl groups having 1 to 20 carbon atoms. More preferred suchunsubstituted alkyl groups have from 1 to 10 carbon atoms while evenmore preferred such groups have from 1 to 6 carbon atoms. Most preferredunsubstituted alkyl groups include straight and branched chain alkylgroups having from 1 to 3 carbon atoms and include methyl, ethyl,propyl, and —CH(CH₃)₂.

The phrase “cycloalkyl” includes cyclic alkyl groups such ascyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, andcyclooctyl and such rings substituted with straight and branched chainalkyl groups as defined above. The phrase also includes polycyclic alkylgroups such as, but not limited to, adamantyl norbornyl, andbicyclo[2.2.2]octyl and such rings substituted with straight andbranched chain alkyl groups as defined above. Thus, the phraseunsubstituted alkyl groups includes primary alkyl groups, secondaryalkyl groups, and tertiary alkyl groups. Unsubstituted alkyl groups maybe bonded to one or more carbon atom(s), oxygen atom(s), nitrogenatom(s), and/or sulfur atom(s) in the parent compound.

The phrase “cycloalkenyl” includes cyclic alkyl groups containing atleast one double bond between two ring carbons such as cyclobutene, andcyclopentene, and cyclohexene. Preferred cycloalkenyl groups have fourto six carbon atoms.

The phrase “substituted alkyl” refers to an unsubstituted alkyl group asdefined above in which one or more bonds to a carbon(s) or hydrogen(s)are replaced by a bond to non-hydrogen and non-carbon atoms such as, butnot limited to, a halogen atom in halides such as F, Cl, Br, and I; aphosphorus atom in groups such as phosphate and dialkylalkylphosphonate; oxygen atom in groups such as hydroxyl groups, alkoxygroups, aryloxy groups, and ester groups; a sulfur atom in groups suchas thiol groups, alkyl and aryl sulfide groups, sulfone groups, sulfonylgroups, and sulfoxide groups; a nitrogen atom in groups such as amines,amides, alkylamines, dialkylamines, arylamines, alkylarylamines,diarylamines, N-oxides, imides, and enamines; a silicon atom in groupssuch as in trialkylsilyl groups, dialkylarylsilyl groups,alkyldiarylsilyl groups, and triarylsilyl groups; and other heteroatomsin various other groups. Substituted alkyl groups also include groups inwhich one or more bonds to a carbon(s) or hydrogen(s) atom is replacedby a bond to a heteroatom such as oxygen in carbonyl, carboxyl, andester groups; nitrogen in groups such as imines, oximes, hydrazones, andnitriles. Preferred substituted alkyl groups include, among others,alkyl groups in which one or more bonds to a carbon or hydrogen atomis/are replaced by one or more bonds to fluorine atoms. One example of asubstituted alkyl group is the trifluoromethyl group and other alkylgroups that contain the trifluoromethyl group. Other alkyl groupsinclude those in which one or more bonds to a carbon or hydrogen atom isreplaced by a bond to an oxygen atom such that the substituted alkylgroup contains a hydroxyl, alkoxy, aryloxy group, or heterocyclyloxygroup. Still other alkyl groups include alkyl groups that have an amine,alkylamine, dialkylamine, arylamine, (alkyl)(aryl)amine, diarylamine,heterocyclylamine, (alkyl)(heterocyclyl)amine,(aryl)(heterocyclyl)amine, or diheterocyclylamine group.

The term “alkoxy” as used herein refers to RO— wherein R, for example,is alkyl such as lower alkyl defined above. Representative examples oflower alkyl alkoxy groups include methoxy, ethoxy, t-butoxy and thelike.

The term “substituted alkoxy” as used herein refers to RO—, where R is,for example, an alkyl substituted, for example, with a halogen. RO isfor example OCF₃. Another example of substituted alkoxy is arylalkoxy.

The term “alkenyl” as used herein refers to a branched or straight chaingroups comprising two to twenty carbon atoms that also comprise one ormore carbon-carbon double bonds. Representative alkenyl groups includeprenyl, 2-propenyl (i.e., allyl), 3-methyl-2-butenyl,3,7-dimethyl-2,6-octadienyl, 4,8-dimethyl-3,7-nonadienyl,3,7,11-trimethyl-2,6,10-dodecatrienyl and the like.

The term “substituted alkenyl” as used herein refers to alkenyl groupsthat are substituted, for example, diethyl hex-5-enylphosphonate, andothers with an alkyl or substituted alkyl group such as dialkylphosphate or an ester such as an acetate ester.

The term “dialkyl amino” as used herein refers to an amino groupsubstituted with two alkyl groups such as C₁₋₂₀ alkyl groups.

The term “substituted dialkyl amino” as used herein refers to adialkylamino substituted, for example, with a carboxylic acid, ester,hydroxy or alkoxy.

The term “hydroxyalkylthio” as used herein refers to a thio radical towhich is appended a hydroxyalkyl group, where the alkyl is for examplelower alkyl. An example is hydroxyethylthio, —SCH₂CH₂OH.

The term “N-alkylsulfonamide” as used herein refers to the group—SO₂NHalkyl, where alkyl is, for example, octyl.

The term “alkynyl” as used herein refers to a branched or straight chaincomprising two to twenty carbon atoms that also comprises one or morecarbon-carbon triple bonds. Representative alkynyl groups includeethynyl, 2-propynyl (propargyl), 1-propynyl and the like. Preferredalkynyl groups have two to six carbon atoms.

The phrase “aryl” refers to aryl groups that do not contain heteroatoms.Thus the phrase includes, but is not limited to, groups such as phenyl,biphenyl, anthracenyl, naphthenyl by way of example. Although the phrase“unsubstituted aryl” includes groups containing condensed rings such asnaphthalene, it does not include aryl groups that have other groups suchas alkyl or halo groups bonded to one of the ring members, as arylgroups such as tolyl are considered herein to be substituted aryl groupsas described below. A preferred unsubstituted aryl group is phenyl.Unsubstituted aryl groups may be bonded to one or more carbon atom(s),oxygen atom(s), nitrogen atom(s), and/or sulfur atom(s) in the parentcompound, however.

The phrase “substituted aryl group” has the same meaning with respect toaryl groups that substituted alkyl groups had with respect to alkylgroups. However, a substituted aryl group also includes aryl groups inwhich one of the aromatic carbons is bonded to one of the non-carbon ornon-hydrogen atoms described above and also includes aryl groups inwhich one or more aromatic carbons of the aryl group is bonded to asubstituted and/or unsubstituted alkyl, alkenyl, or alkynyl group asdefined herein. This includes bonding arrangements in which two carbonatoms of an aryl group are bonded to two atoms of an alkyl, alkenyl, oralkynyl group to define a fused ring system (e.g. dihydronaphthyl ortetrahydronaphthyl). Thus, the phrase “substituted aryl” includes, butis not limited to tolyl, and hydroxyphenyl among others.

The term “arylalkyl” as used herein refers to a loweralkyl radical towhich is appended an aryl group. Representative arylalkyl groups includebenzyl, phenylethyl, hydroxybenzyl, fluorobenzyl, fluorophenylethyl andthe like.

The term “arylalkylaryl” as used herein refers to an arylalkyl group aspreviously defined appended to an aryl group. Representativearylalkylaryl groups include 4-benzylphenyl, 3-benzylphenyl,4-phenethylphenyl and the like.

The term “arylaryl” as used herein refers to an aryl group as previouslydefined that is appended to an aryl group. Representative arylarylgroups include biphenyl, 4-(1-naphthyl)phenyl, 4-(2-naphthyl)phenyl andthe like.

The term “aryloxy” as used herein refers to RO— wherein R is an arylgroup. Representative arylalkoxy group include benzyloxy, phenylethoxyand the like.

The term “arylalkoxy” as used herein refers to a lower alkoxy radical towhich is appended an aryl group. Representative arylalkoxy group includebenzyloxy, phenylethoxy and the like.

The term “aryloxyaryl” as used herein refers to an aryl radical to whichis appended an aryloxy group. Representative aryloxyaryl groups include4-phenoxyphenyl, 3-phenoxyphenyl, 4-phenoxy-1-naphthyl,3-phenoxy-1-naphthyl and the like.

The term “aryloxyarylalkyl” as used herein refers to an arylalkylradical to which is appended an aryloxy group. Representativearyloxyarylalkyl groups include 4-phenoxyphenylmethyl,3-phenoxyphenylmethyl, 4-phenoxyphenylethyl, 3-phenoxy-phenylethyl andthe like.

The term “arylalkoxyaryl” as used herein refers to an aryl radical towhich is appended an arylalkoxy group. Representative arylalkoxyarylgroups include 4-benzyloxy]phenyl, 3-benzyloxyphenyl and the like.

The term “arylalkoxyarylalkyl” as used herein refers to an arylalkylradical to which is appended an arylalkoxy group. Representativearylalkoxyarylalkyl groups include 4-benzyloxy]benzyl, 3-benzyloxybenzyland the like.

The term “cycloalkylalkyl” as used herein refers to a loweralkyl radicalto which is appended a cycloalkyl group. Representative examples ofcycloalkylalkyl include cyclopropylmethyl, cyclohexylmethyl,2-(cyclopropyl)ethyl and the like.

The term “halogen” refers to iodine, bromine, chlorine or fluorine;“halo” as used herein refers to iodo, bromo, chloro or fluoro.

The term “haloalkyl” as used herein refers to a lower alkyl radical, asdefined above, bearing at least one halogen substituent, for example,chloromethyl, fluoroethyl or trifluoromethyl and the like.

The phrase “heterocyclyl” refers to both aromatic and nonaromatic ringcompounds including monocyclic, bicyclic, and polycyclic ring compoundssuch as, but not limited to, quinuclidyl, containing 3 or more ringmembers of which one or more is a heteroatom such as, but not limitedto, N, O, and S. Although the phrase “unsubstituted heterocyclyl”includes condensed heterocyclic rings such as benzimidazolyl, it doesnot include heterocyclyl groups that have other groups such as alkyl orhalo groups bonded to one of the ring members as compounds such as2-methylbenzimidazolyl are substituted heterocyclyl groups. Examples ofheterocyclyl groups include, but are not limited to: unsaturated 3 to 8membered rings containing 1 to 4 nitrogen atoms such as, but not limitedto pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, pyridyl, dihydropyridyl,pyrimidyl, pyrazinyl, pyridazinyl, triazolyl (e.g. 4H-1,2,4-triazolyl,1H-1,2,3-triazolyl, 2H-1,2,3-triazolyl etc.), tetrazolyl, (e.g.1H-tetrazolyl, 2H tetrazolyl, etc.); saturated 3 to 8 membered ringscontaining 1 to 4 nitrogen atoms such as, but not limited to,pyrrolidinyl, imidazolidinyl, piperidinyl, piperazinyl; condensedunsaturated heterocyclic groups containing 1 to 4 nitrogen atoms suchas, but not limited to, indolyl, isoindolyl, indolinyl, indolizinyl,benzimidazolyl, quinolyl, isoquinolyl, indazolyl, benzotriazolyl;unsaturated 3 to 8 membered rings containing 1 to 2 oxygen atoms and 1to 3 nitrogen atoms such as, but not limited to, oxazolyl, isoxazolyl,oxadiazolyl (e.g. 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl,1,2,5-oxadiazolyl, etc.); saturated 3 to 8 membered rings containing 1to 2 oxygen atoms and 1 to 3 nitrogen atoms such as, but not limited to,morpholinyl; unsaturated condensed heterocyclic groups containing 1 to 2oxygen atoms and 1 to 3 nitrogen atoms, for example, benzoxazolyl,benzoxadiazolyl, benzoxazinyl (e.g. 2H-1,4-benzoxazinyl etc.);unsaturated 3 to 8 membered rings containing 1 to 3 sulfur atoms and 1to 3 nitrogen atoms such as, but not limited to, thiazolyl,isothiazolyl, thiadiazolyl (e.g. 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl,1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl, etc.); saturated 3 to 8 memberedrings containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms such as,but not limited to, thiazolodinyl; saturated and unsaturated 3 to 8membered rings containing 1 to 2 sulfur atoms such as, but not limitedto, thienyl, dihydrodithiinyl, dihydrodithionyl, tetrahydrothiophene,tetrahydrothiopyran; unsaturated condensed heterocyclic rings containing1 to 2 sulfur atoms and 1 to 3 nitrogen atoms such as, but not limitedto, benzothiazolyl, benzothiadiazolyl, benzothiazinyl (e.g.2H-1,4-benzothiazinyl, etc.), dihydrobenzothiazinyl (e.g.2H-3,4-dihydrobenzothiazinyl, etc.), unsaturated 3 to 8 membered ringscontaining oxygen atoms such as, but not limited to furyl; unsaturatedcondensed heterocyclic rings containing 1 to 2 oxygen atoms such asbenzodioxolyl (e.g. 1,3-benzodioxoyl, etc.); unsaturated 3 to 8 memberedrings containing an oxygen atom and 1 to 2 sulfur atoms such as, but notlimited to, dihydrooxathiinyl; saturated 3 to 8 membered ringscontaining 1 to 2 oxygen atoms and 1 to 2 sulfur atoms such as1,4-oxathiane; unsaturated condensed rings containing 1 to 2 sulfuratoms such as benzothienyl, benzodithiinyl; and unsaturated condensedheterocyclic rings containing an oxygen atom and 1 to 2 oxygen atomssuch as benzoxathiinyl. Heterocyclyl group also include those describedabove in which one or more S atoms in the ring is double-bonded to oneor two oxygen atoms (sulfoxides and sulfones). For example, heterocyclylgroups include tetrahydrothiophene, tetrahydrothiophene oxide, andtetrahydrothiophene 1,1-dioxide. Preferred heterocyclyl groups contain 5or 6 ring members. More preferred heterocyclyl groups includemorpholine, piperazine, piperidine, pyrrolidine, imidazole, pyrazole,1,2,3-triazole, 1,2,4-triazole, tetrazole, thiomorpholine,thiomorpholine in which the S atom of the thiomorpholine is bonded toone or more O atoms, pyrrole, homopiperazine, oxazolidin-2-one,pyrrolidin-2-one, oxazole, quinuclidine, thiazole, isoxazole, furan, andtetrahydrofuran.

The phrase “substituted heterocyclyl” refers to an heterocyclyl group asdefined above in which one of the ring members is bonded to anon-hydrogen atom such as described above with respect to substitutedalkyl groups and substituted aryl groups. Examples, include, but are notlimited to, 2-methylbenzimidazolyl, 5-methylbenzimidazolyl,5-chlorobenzthiazolyl, 1-methyl piperazinyl, t-butyloxycarbonyl, and2-chloropyridyl among others.

“Substituted” refers to the definite replacement of hydrogen with one ormore monovalent or divalent radicals. Suitable substitution groupsinclude, those described herein for particular groups, as well ashydroxyl, nitro, amino, imino, cyano, halo, thio, thioamido, amidino,imidino, oxo, oxamidino, methoxamidino, imidino, guanidino, sulfonamido,carboxyl, formyl, alkyl, substituted alkyl, haloloweralkyl, loweralkoxy,haloloweralkoxy, loweralkoxyalkyl, alkylcarbonyl, arylcarbonyl,aralkylcarbonyl, heteroarylcarbonyl, heteroaralkylcarbonyl, alkylthio,aminoalkyl, cyanoalkyl, benzyl, pyridyl, pyrazolyl, pyrrole, thiophene,imidazolyl, and the like.

By overall atom length, e.g., in the context of a linker of theinvention, is meant the number of atoms in a single chain, not countingsubstituents. For instance, —CH₂— counts as one atom with respect tooverall linker length, —CH₂CH₂O— counts as 3 atoms in length, and anon-linear group such as a phenyl ring counts as 4 atoms in length.

“Substantially” or “essentially” means nearly totally or completely, forinstance, 95% or greater of some given quantity.

“Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.

The terms “polypeptide,” and “protein” are used interchangeably hereinto denote any polymer of amino acid residues. The terms encompasspeptides, oligopeptides, dimers, multimers, and the like. Suchpolypeptides can be derived from natural sources or can be synthesizedor recombinantly produced. The terms also include postexpressionmodifications of the polypeptide, for example, glycosylation,acetylation, phosphorylation, etc.

A polypeptide as defined herein is generally made up of the 20 naturalamino acids Ala (A), Arg (R), Asn (N), Asp (D), Cys (C), Gln (O), Glu(E), Gly (G), H is (H), Ile (I), Leu (L), Lys (K), Met (M), Phe (F), Pro(P), Ser (S), Thr (T), Trp (W), Tyr (Y) and Val (V) and may also includeany of the several known amino acid analogs, both naturally occurringand synthesized analogs, such as but not limited to homoisoleucine,asaleucine, 2-(methylenecyclopropyl)glycine, S-methylcysteine,S-(prop-1-enyl)cysteine, homoserine, ornithine, norleucine, norvaline,homoarginine, 3-(3-carboxyphenyl)alanine, cyclohexylalanine, mimosine,pipecolic acid, 4-methylglutamic acid, canavanine, 2,3-diaminopropionicacid, and the like. Further examples of polypeptide agents that willfind use in the present invention are set forth below.

By “geometry” or “tertiary structure” of a polypeptide or protein ismeant the overall 3-D configuration of the protein. As described herein,the geometry can be determined, for example, by crystallography studiesor by using various programs or algorithms that predict the geometrybased on interactions between the amino acids making up the primary andsecondary structures.

By “wild type” or “native” polypeptide, polypeptide agent or polypeptidedrug, is meant a naturally occurring polypeptide sequence, and itscorresponding secondary structure. An “isolated” or “purified” proteinor polypeptide is a protein that is separate and discrete from a wholeorganism with which the protein is normally associated in nature. It isapparent that the term denotes proteins of various levels of purity.Typically, a composition containing a purified protein will be one inwhich at least about 35%, preferably at least about 40-50%, morepreferably, at least about 75-85%, and most preferably at least about90% or more, of the total protein in the composition will be the proteinin question.

By “Env polypeptide” is meant a molecule derived from an envelopeprotein, preferably from HIV Env. The envelope protein of HIV-1 is aglycoprotein of about 160 kd (gp160). During virus infection of the hostcell, gp160 is cleaved by host cell proteases to form gp120 and theintegral membrane protein, gp41. The gp41 portion is anchored in (andspans) the membrane bilayer of virion, while the gp120 segment protrudesinto the surrounding environment. As there is no covalent attachmentbetween gp120 and gp41, free gp120 is released from the surface ofvirions and infected cells. Env polypeptides may also include gp 140polypeptides. Env polypeptides can exist as monomers, dimers ormultimers.

By a “gp120 polypeptide” is meant a molecule derived from a gp120 regionof the Env polypeptide. Preferably, the gp120 polypeptide is derivedfrom HIV Env. The primary amino acid sequence of gp120 is approximately511 amino acids, with a polypeptide core of about 60,000 Daltons. Thepolypeptide is extensively modified by N-linked glycosylation toincrease the apparent molecular weight of the molecule to 120,000Daltons. The amino acid sequence of gp120 contains five relativelyconserved domains interspersed with five hypervariable domains. Thepositions of the 18 cysteine residues in the gp120 primary sequence ofthe HIV-1_(HXB-2) (hereinafter “HXB-2”) strain, and the positions of 13of the approximately 24 N-linked glycosylation sites in the gp120sequence are common to most, if not all, gp120 sequences. Thehypervariable domains contain extensive amino acid substitutions,insertions and deletions. Despite this variation, most, if not all,gp120 sequences preserve the virus's ability to bind to the viralreceptor CD4. A “gp120 polypeptide” includes both single subunits and/ormultimers.

Env polypeptides (e.g., gp120, gp140 and gp160) include a “bridgingsheet” comprised of 4 anti-parallel β-strands (β-2, β-3, β-20 and β-21)that form a β-sheet.

Extruding from one pair of the β-strands (β-2 and 3-3) are two loops, V1and V2. The 3-2 sheet occurs at approximately amino acid residue 119(Cys) to amino acid residue 123 (Thr) while 3-3 occurs at approximatelyamino acid residue 199 (Ser) to amino acid residue 201 (Ile), relativeto HXB-2. The “V1/V2 region” occurs at approximately amino acidpositions 126 (Cys) to residue 196 (Cys), relative to HXB-2. (see, e.g.,Wyatt et al. (1995) J. Virol. 69:5723-5733; Stamatatos et al. (1998) J.Virol. 72:7840-7845). Extruding from the second pair of β-strands (β-20and β-21) is a “small-loop” structure, also referred to herein as “thebridging sheet small loop.” In HXB-2, 3-20 extends from about amino acidresidue 422 (Gln) to amino acid residue 426 (Met) while β-21 extendsfrom about amino acid residue 430 (Val) to amino acid residue 435 (Tyr).In variant SF162, the Met-426 is an Arg (R) residue. The “small loop”extends from about amino acid residue 427 (Trp) through 429 (Lys),relative to HXB-2. Alignment of the amino acid sequences of Envpolypeptide gp160 of any HIV variant can be determined relative to othervariants, such as HXB-2, as described for example, in WO 00/39303.

Furthermore, an “Env polypeptide” or “gp120 polypeptide” as definedherein is not limited to a polypeptide having the exact sequencedescribed herein. Indeed, the HIV genome is in a state of constant fluxand contains several variable domains that exhibit relatively highdegrees of variability between isolates. It is readily apparent that theterms encompass Env (e.g., gp120) polypeptides from any of theidentified HIV isolates, as well as newly identified isolates, andsubtypes of these isolates. Descriptions of structural features aregiven herein with reference to HXB-2. One of ordinary skill in the artin view of the teachings of the present disclosure and the art candetermine corresponding regions in other HIV variants (e.g., isolatesHIV_(IIIb), HIV_(SF2), HIV-1_(SF162), HIV-1_(SF170), HIV_(LAV),HIV_(LAI), HIV_(MN), HIV-1_(CM235), HIV-1_(US4), other HIV-1 strainsfrom diverse subtypes (e.g., subtypes, A through G, and O), HIV-2strains and diverse subtypes (e.g., HIV-2_(UC1) and HIV-2_(UC2)), andsimian immunodeficiency virus (SIV). (See, e.g., Virology, 3rd Edition(W. K. Joklik ed. 1988); Fundamental Virology, 2nd Edition (B. N. Fieldsand D. M. Knipe, eds. 1991); Virology, 3rd Edition (Fields, B N, D MKnipe, P M Howley, Editors, 1996, Lippincott-Raven, Philadelphia, Pa.;for a description of these and other related viruses), using forexample, sequence comparison programs (e.g., BLAST and others describedherein) or identification and alignment of structural features (e.g., aprogram such as the “ALB” program described herein that can identifyβ-sheet regions). The actual amino acid sequences of the modified Envpolypeptides can be based on any HIV variant.

Additionally, the term “Env polypeptide” (e.g., “gp120 polypeptide”)encompasses proteins that include additional modifications to the nativesequence, such as additional internal deletions, additions andsubstitutions. These modifications may be deliberate, as throughsite-directed mutagenesis, or may be accidental, such as throughnaturally occurring mutational events. Thus, for example, if the Envpolypeptide is to be used in vaccine compositions, the modificationsmust be such that immunological activity (i.e., the ability to elicit anantibody response to the polypeptide) is not lost. Similarly, if thepolypeptides are to be used for diagnostic purposes, such capabilitymust be retained.

The term “modified Env polypeptide” is an Env polypeptide (e.g., gp120as defined above) refers to an Env polypeptide that has been complexedto a CD4 mimetic. The Env polypeptide may optionally be modified inother ways, for example in the variable regions V1 and V2. The Envpolypeptide may be monomeric or oligomeric. Generally, complexed Env(e.g., gp120) polypeptides result in exposure of epitopes in or near theCD4 binding site, while allowing correct folding (e.g., correctgeometry) of the Env polypeptide. Additionally, modifications (e.g.,truncations) to the V1 and V2 loop regions may also be made. Althoughnot all possible V1/V2 modifications have been exemplified herein, it isto be understood that other disrupting modifications are alsoencompassed by the present invention.

By “binding” is meant the ability of a CD4 mimetic (e.g., peptoid, smallmolecule, peptoid small molecule conjugate) to specifically interactwith an Env polypeptide such that interaction results in aconformational change in the Env polypeptide that leads to exposure ofEnv epitopes to which neutralizing antibodies are more readilygenerated.

A polypeptide (e.g., gp120 or other Env polypeptide) is produced“intracellularly” when it is found within the cell, either associatedwith components of the cell, such as in association with the endoplasmicreticulum (ER) or the Golgi Apparatus, or when it is present in thesoluble cellular fraction. The gp120 and other Env polypeptides of thepresent invention may also be secreted into growth medium so long assufficient amounts of the polypeptides remain present within the cellsuch that they can be purified from cell lysates using techniquesdescribed herein.

An “immunogenic” gp120 or other Env protein is a molecule that includesat least one epitope such that the molecule is capable of eithereliciting an immunological reaction in an individual to which theprotein is administered or, in the diagnostic context, is capable ofreacting with antibodies directed against the HIV in question.

By “epitope” is meant a site on an antigen to which specific B cellsand/or T cells respond, rendering the molecule including such an epitopecapable of eliciting an immunological reaction or capable of reactingwith HIV antibodies present in a biological sample. The term is alsoused interchangeably with “antigenic determinant” or “antigenicdeterminant site.” An epitope can comprise 3 or more amino acids in aspatial conformation unique to the epitope. Generally, an epitopeconsists of at least 5 such amino acids and, more usually, consists ofat least 8-10 such amino acids. Methods of determining spatialconformation of amino acids are known in the art and include, forexample, x-ray crystallography and 2-dimensional nuclear magneticresonance. Furthermore, the identification of epitopes in a givenprotein is readily accomplished using techniques well known in the art,such as by the use of hydrophobicity studies and by site-directedserology. See, also, Geysen et al., Proc. Natl. Acad. Sci. USA (1984)81:3998-4002 (general method of rapidly synthesizing peptides todetermine the location of immunogenic epitopes in a given antigen); U.S.Pat. No. 4,708,871 (procedures for identifying and chemicallysynthesizing epitopes of antigens); and Geysen et al., MolecularImmunology (1986) 23:709-715 (technique for identifying peptides withhigh affinity for a given antibody). Antibodies that recognize the sameepitope can be identified in a simple immunoassay showing the ability ofone antibody to block the binding of another antibody to a targetantigen. A “cryptic epitope” refers generally to an epitope that isexposed only in certain conformations of the protein.

A “functional epitope” refers to an epitope that elicits antibody whichprevents or limits HIV infection. In a particular embodiment, theantibodies are neutralizing antibodies. In other embodiments, theantibodies can, e.g., elicit an ADCC response.

An “immunological response” or “immune response” as used herein caninduce the development in the subject of a humoral and/or a cellularimmune response to the Env (e.g., gp120) polypeptide when thepolypeptide is present in a vaccine composition. Antibodies elicited inan immune response may also neutralize infectivity, and/or mediateantibody-complement or antibody dependent cell cytotoxicity to provideprotection to an immunized host. Immunological reactivity may bedetermined in standard immunoassays, such as a competition assays, wellknown in the art.

The term “antibody” as used herein includes antibodies obtained fromboth polyclonal and monoclonal preparations, as well as, the following:(i) hybrid (chimeric) antibody molecules (see, for example, Winter etal. (1991) Nature 349:293-299; and U.S. Pat. No. 4,816,567); (ii)F(ab′)2 and F(ab) fragments; (iii) Fv molecules (noncovalentheterodimers, see, for example, Inbar et al. (1972) Proc. Natl. Acad.Sci. USA 69:2659-2662; and Ehrlich et al. (1980) Biochem 19:4091-4096);(iv) single-chain Fv molecules (sFv) (see, for example, Huston et al.(1988) Proc. Natl. Acad. Sci. USA 85:5879-5883); (v) dimeric andtrimeric antibody fragment constructs; (vi) humanized antibody molecules(see, for example, Riechmann et al. (1988) Nature 332:323-327; Verhoeyanet al. (1988) Science 239:1534-1536; and U.K. Patent Publication No. GB2,276,169, published 21 Sep. 1994); (vii) Mini-antibodies or minibodies(i.e., sFv polypeptide chains that include oligomerization domains attheir C-termini, separated from the sFv by a hinge region; see, e.g.,Pack et al. (1992) Biochem 31:1579-1584; Cumber et al. (1992) J.Immunology 149B:120-126); (vii) human antibodies; and, (viii) anyfunctional fragments obtained from such molecules, wherein suchfragments retain specific-binding properties of the parent antibodymolecule.

Thus, the term “antibody” refers to a polypeptide or group ofpolypeptides that comprise at least one antigen-binding site. An“antigen binding site” is formed from the folding of the variabledomains of an antibody molecule(s) to form three-dimensional bindingsites with an internal surface shape and charge distributioncomplementary to the features of an epitope of an antigen, which allowsspecific binding to form an antibody-antigen complex. An antigen-bindingsite may be formed from a heavy- and/or light-chain domain (VH and VL,respectively), which form hypervariable loops that contribute to antigenbinding. The term “antibody” includes, without limitation, polyclonalantibodies, monoclonal antibodies, chimeric antibodies, alteredantibodies, univalent antibodies, Fab proteins, and single-domainantibodies. In many cases, the binding phenomena of antibodies toantigens is equivalent to other ligand/anti-ligand binding.

If polyclonal antibodies are desired, a selected mammal (e.g., mouse,rabbit, goat, horse, etc.) is immunized with an immunogenic polypeptidebearing an HCV epitope(s). Serum from the immunized animal is collectedand treated according to known procedures. If serum containingpolyclonal antibodies to an HCV epitope contains antibodies to otherantigens, the polyclonal antibodies can be purified by immunoaffinitychromatography. Techniques for producing and processing polyclonalantisera are known in the art, see for example, Mayer and Walker, eds.(1987) IMMUNOCHEMICAL METHODS IN CELL AND MOLECULAR BIOLOGY (AcademicPress, London).

One skilled in the art can also readily produce monoclonal antibodiesdirected against HIV epitopes. The general methodology for makingmonoclonal antibodies by hybridomas is well known. Immortalantibody-producing cell lines can be created by cell fusion, and also byother techniques such as direct transformation of B lymphocytes withoncogenic DNA, or transfection with Epstein-Barr virus. See, e.g., M.Schreier et al. (1980) HYBRIDOMA TECHNIQUES; Hammerling et al. (1981),MONOCLONAL ANTIBODIES AND T-CELL HYBRIDOMAS; Kennett et al. (1980)MONOCLONAL ANTIBODIES; see also, U.S. Pat. Nos. 4,341,761; 4,399,121;4,427,783; 4,444,887; 4,466,917; 4,472,500; 4,491,632; and 4,493,890.Panels of monoclonal antibodies produced against HCV epitopes can bescreened for various properties; i.e., for isotype, epitope affinity,etc. As used herein, a “single domain antibody” (dAb) is an antibodythat is comprised of an HL domain, which binds specifically with adesignated antigen. An Ab does not contain a VL domain, but may containother antigen binding domains known to exist to antibodies, for example,the kappa and lambda domains. Methods for preparing dabs are known inthe art. See, for example, Ward et al, Nature 341: 544 (1989).

Antibodies can also be comprised of VH and VL domains, as well as otherknown antigen binding domains. Examples of these types of antibodies andmethods for their preparation and known in the art (see, e.g., U.S. Pat.No. 4,816,467), and include the following. For example, “vertebrateantibodies” refers to antibodies that are tetramers or aggregatesthereof, comprising light and heavy chains which are usually aggregatedin a “Y” configuration and which may or may not have covalent linkagesbetween the chains. In vertebrate antibodies, the amino acid sequencesof the chains are homologous with those sequences found in antibodiesproduced in vertebrates, whether in situ or in vitro (for example, inhybridomas). Vertebrate antibodies include, for example, purifiedpolyclonal antibodies and monoclonal antibodies, methods for thepreparation of which are described infra.

“Hybrid antibodies” are antibodies where chains are separatelyhomologous with reference to mammalian antibody chains and representnovel assemblies of them, so that two different antigens areprecipitable by the tetramer or aggregate. In hybrid antibodies, onepair of heavy and light chains are homologous to those found in anantibody raised against a first antigen, while a second pair of chainsare homologous to those found in an antibody raised against a secondantibody. This results in the property of “divalence”, i.e., the abilityto bind two antigens simultaneously. Such hybrids can also be formedusing chimeric chains, as set forth below.

“Chimeric antibodies” refers to antibodies in which the heavy and/orlight chains are fusion proteins. Typically, one portion of the aminoacid sequences of the chain is homologous to corresponding sequences inan antibody derived from a particular species or a particular class,while the remaining segment of the chain is homologous to the sequencesderived from another species and/or class. Usually, the variable regionof both light and heavy chains mimics the variable regions or antibodiesderived from one species of vertebrates, while the constant portions arehomologous to the sequences in the antibodies derived from anotherspecies of vertebrates. However, the definition is not limited to thisparticular example. Also included is any antibody in which either orboth of the heavy or light chains are composed of combinations ofsequences mimicking the sequences in antibodies of different sources,whether these sources be from differing classes or different species oforigin, and whether or not the fusion point is at the variable/constantboundary. Thus, it is possible to produce antibodies in which neitherthe constant nor the variable region mimic know antibody sequences. Itthen becomes possible, for example, to construct antibodies whosevariable region has a higher specific affinity for a particular antigen,or whose constant region can elicit enhanced complement fixation, or tomake other improvements in properties possessed by a particular constantregion.

Another example is “altered antibodies”, which refers to antibodies inwhich the naturally occurring amino acid sequence in a vertebrateantibody has been varies. Utilizing recombinant DNA techniques,antibodies can be redesigned to obtain desired characteristics. Thepossible variations are many, and range from the changing of one or moreamino acids to the complete redesign of a region, for example, theconstant region. Changes in the constant region, in general, to attaindesired cellular process characteristics, e.g., changes in complementfixation, interaction with membranes, and other effector functions.Changes in the variable region can be made to alter antigen-bindingcharacteristics. The antibody can also be engineered to aid the specificdelivery of a molecule or substance to a specific cell or tissue site.The desired alterations can be made by known techniques in molecularbiology, e.g., recombinant techniques, site-directed mutagenesis, etc.

Yet another example are “univalent antibodies”, which are aggregatescomprised of a heavy-chain/light-chain dimer bound to the Fc (i.e.,stem) region of a second heavy chain. This type of antibody escapesantigenic modulation. See, e.g., Glennie et al. Nature 295: 712 (1982).Included also within the definition of antibodies are “Fab” fragments ofantibodies. The “Fab” region refers to those portions of the heavy andlight chains which are roughly equivalent, or analogous, to thesequences which comprise the branch portion of the heavy and lightchains, and which have been shown to exhibit immunological binding to aspecified antigen, but which lack the effector Fc portion. “Fab”includes aggregates of one heavy and one light chain (commonly known asFab′), as well as tetramers containing the 2H and 2L chains (referred toas F(ab)₂), which are capable of selectively reacting with a designatedantigen or antigen family. Fab antibodies can be divided into subsetsanalogous to those described above, i.e., “vertebrate Fab”, “hybridFab”, “chimeric Fab”, and “altered Fab”. Methods of producing Fabfragments of antibodies are known within the art and include, forexample, proteolysis, and synthesis by recombinant techniques.

“Antigen-antibody complex” refers to the complex formed by an antibodythat is specifically bound to an epitope on an antigen.

Techniques for determining amino acid sequence “similarity” are wellknown in the art. In general, “similarity” means the exact amino acid toamino acid comparison of two or more polypeptides at the appropriateplace, where amino acids are identical or possess similar chemicaland/or physical properties such as charge or hydrophobicity. A so-termed“percent similarity” then can be determined between the comparedpolypeptide sequences. Techniques for determining nucleic acid and aminoacid sequence identity also are well known in the art and includedetermining the nucleotide sequence of the mRNA for that gene (usuallyvia a cDNA intermediate) and determining the amino acid sequence encodedthereby, and comparing this to a second amino acid sequence. In general,“identity” refers to an exact nucleotide to nucleotide or amino acid toamino acid correspondence of two polynucleotides or polypeptidesequences, respectively.

Computer programs are available to determine the likelihood of certainpolypeptides to form structures such as β-sheets. One such program,described herein, is the “ALB” program for protein and polypeptidesecondary structure calculation and predication. In addition, secondaryprotein structure can be predicted from the primary amino acid sequence,for example using protein crystal structure and aligning the proteinsequence related to the crystal structure (e.g., using MolecularOperating Environment (MOE) programs available from the ChemicalComputing Group Inc., Montreal, P.Q., Canada). Other methods ofpredicting secondary structures are described, for example, in Garnieret al. (1996) Methods Enzymol. 266:540-553; Geourjon et al. (1995)Comput. Applic. Biosci. 11:681-684; Levin (1997) Protein Eng.10:771-776; and Rost et al. (1993) J. Molec. Biol. 232:584-599.

By “vertebrate subject” is meant any member of the subphylum chordata,including, without limitation, humans and other primates, includingnon-human primates such as chimpanzees and other apes and monkeyspecies; farm animals such as cattle, sheep, pigs, goats and horses;domestic mammals such as dogs and cats; laboratory animals includingrodents such as mice, rats and guinea pigs; birds, including domestic,wild and game birds such as chickens, turkeys and other gallinaceousbirds, ducks, geese, and the like. The term does not denote a particularage. Thus, both adult and newborn individuals are intended to becovered.

As used herein, a “biological sample” refers to a sample of tissue orfluid isolated from an individual, including but not limited to, forexample, blood, plasma, serum, fecal matter, urine, bone marrow, bile,spinal fluid, lymph fluid, samples of the skin, external secretions ofthe skin, respiratory, intestinal, and genitourinary tracts, samplesderived from the gastric epithelium and gastric mucosa, tears, saliva,milk, blood cells, organs, biopsies and also samples of in vitro cellculture constituents including but not limited to conditioned mediaresulting from the growth of cells and tissues in culture medium, e.g.,recombinant cells, and cell components.

The terms “label” and “detectable label” refer to a molecule capable ofdetection, including, but not limited to, radioactive isotopes,fluorescers, chemiluminescers, enzymes, enzyme substrates, enzymecofactors, enzyme inhibitors, chromophores, dyes, metal ions, metalsols, ligands (e.g., biotin or haptens) and the like. The term“fluorescer” refers to a substance or a portion thereof that is capableof exhibiting fluorescence in the detectable range. Particular examplesof labels that may be used with the invention include, but are notlimited to fluorescein, rhodamine, dansyl, umbelliferone, Texas red,luminol, acradimum esters, NADPH, beta-galactosidase, horseradishperoxidase, glucose oxidase, alkaline phosphatase and urease.

OVERVIEW

The present invention is directed to CD4 mimetics, complexes of Env andCD4 mimetics (Env-CD4 complexes), antibodies to these complexes and theuse of compositions comprising a CD4 mimetic and an Env. Without beingbound by a particular theory, it appears that it has been difficult togenerate immunological responses against Env because the CD4 bindingsite (and/or the CCR binding site) is buried between the outer domain,the inner domain, and the V1/V2 domains of Env. Thus, although deletionof the V1/V2 domain may render the virus more susceptible toneutralization by monoclonal antibody directed to the CD4 site, theconformation of Env prior to CD4 binding may prevent an antibodyresponse. Thus, the present invention provides CD4 mimetics, and inparticular, small molecule CD4 mimetics, that bind to Env and appear tocause a conformational change in Env that exposes one or more epitopes(e.g., “cryptic” or “inducible” epitopes) in or near the CD4 bindingsite, which in turn allows the generation of an immune response (e.g., aneutralizing antibody response) to Env.

It is to be understood that various forms of the different embodimentsof the invention, described herein, may be combined.

CD4 Mimetics

CD4 mimetics described herein include peptoids, small molecules, andpeptoid small molecule conjugates that bind to Env polypeptides toinduce a conformational change in the Env protein similar or identicalto the change induced when Env binds to CD4. The amino acid sequence ofCD4 is known and structural studies on CD4 have shown that this moleculeis composed of four extracellular immunoglobulin like domains (threecontaining disulfide linked loops). It is also known that the binding ofgp120 to its receptor (CD4) induces conformational changes in the Envprotein. However only domain 1 (D1) of CD4 is critical for itsinteraction with gp120 (Arthos et al. (1989) Cell 57(3):469-81; Trunehet al. (1991) J Biol Chem 266(9):5942-8). Mutational analyses, antibodycompetition experiments combined with the knowledge of three-dimensionalstructure of CD4 have shown that a region homologous to complementaritydetermining region 2 (CDR2) of immunoglobulin in D1 plays a major rolein gp120 binding (Ryu et al. (1994) Structure 2(1):59-74, Sullivan etal. (1998) J Virol 72(8):6332-6338).

Indeed, structure resolution of gp120:CD4 complex confirmed that theCDR2-like loop of CD4 is central in CD4-gp120 interaction (Choe &Sodroski (1992) J Acquir Immune Defic Syndr 5(2):204-10, Gizachew et al.(1998) Biochemistry 37(30):10616-25). In the complex CD4 Phe-43 sidechain plugs the entrance of a deep cavity in gp120 and CD4 Arg59, justbehind Phe43, is involved in a double H-bond with Asp-368 in gp120.

Crystallographic structure analysis of gp120, in complex with CD4 andthe Fab portion the neutralizing monoclonal antibody 17b (Thali et al.(1993) J Virol 67(7):3978-88; Kwong et al. (1998) Nature 393:648-659),indicates that a large surface (742 Å2) of the domain D1 of CD4 binds toa large depression (800 A2) on gp120. The CD4 interface is comprised of22 residues, contributing to gp120 binding with mixed hydrophobic,electrostatic, H-bonding interactions. The large size and complexity ofthis interface makes the reproduction of such functional epitope into asmall molecule a challenge, and explains the difficulty in thedevelopment of small molecule inhibitors of gp120-CD4 interaction. Vitaet al. (1998) Biopolymers 47:93-100. However, in spite of the largenumber of residues present in gp120-CD4 interaction surface, studies onhormone-receptor systems showed that only a few residues might dominatethe binding energy at the protein-protein interface. Clackson and Wells(1995) Science 267(5196):383-386.

Upon binding of gp120 to CD4, unique neutralizing epitopes also appearto be exposed, for example the epitope recognized by the monoclonalantibody CG10 (Gershoni et al. (1993) Faseb J 7(12):1185-7). Indeed,while monomeric gp120 protein from lab strains is poorly immunogenicwith regard to eliciting primary isolate neutralizing antibodies(Mascola et al. (1996) J. Infect. Dis. 173:340-348), monoclonalantibodies that appear to recognize certain epitopes that are exposed onthe Env surface once it binds to its CD4 receptor have been shown toneutralize diverse primary isolates. See, e.g., the monoclonal antibodydesignated 17b (Thali et al. (1993) J Virol 67(7):3978-3988). However,cross-clade primary isolate neutralizing antibody responses usingreceptor/co-receptor complexed Env have been attributed to theimmunogenicity of the gp41 fusion domain. Lacasse et al (1999) Science283:357-362.

Additionally, attempts to evaluate gp120-CD4 complexes as potentialvaccine candidate for inducing high avidity and primary isolateneutralizing antibodies have been thwarted by the concern that an immuneresponse could be generated against CD4 itself thereby raisingautoimmune and safety issues. (D'Souza et al. (1997) JInfect Dis.175:1056-62, DeVico et al. (1995) Virology 211(2):583-588).

Thus, the present invention pertains, in part, to CD4 mimetics that bindto HIV Env polypeptides and when bound, induce a conformational changein Env similar to that induced by binding to CD4.

In other embodiments the immunological compositions provided by thepresent invention include small molecule compounds that are known to becompetitive CD4 inhibitors of a gp120 polypeptide (Shaoxing, C. et. al.Design and synthesis of a CD4 β-turn mimetic that inhibits humanimmunodeficiency virus envelope glycoprotein gp120 binding and infectionof human lymphocytes, Proc. Natl. Acad. Sci., (1992), 89, 5872-5876; WO99/24065, WO 03/072028, U.S. Pat. Nos. 6,469,006 and 6,476,034).

In still other embodiments, the present invention provides immunologicalcompositions containing novel small molecule CD4 mimetics. These smallmolecules are commercially available or may be synthesized by one ofskill in the art by adapting the procedures set forth in WO 03/072028,U.S. Pat. No. 6,469,006, and U.S. Pat. No. 6,476,034.

In particular, preferred CD4 mimetics are small molecules having amolecular weight of less than about 2500 daltons. However, moretypically, a small molecule CD4-mimetic in accordance with the inventionwill possess a molecular weight falling within one of the followingranges: less than about 1500, less than about 1000, less than about 900,less than about 800, less than about 700, less than about 600 or lessthan about 500.

Certain small molecule CD4 mimetics of the invention were identified byfirst screening molecules having a degree of structural similarity toCD4M33, a known CD4 mimetic (Martin et al (2003), Nat. Biotechnol.21(7):71-76), as described in detail in Example 1. In examining smallmolecules having the desired features (e.g., the ability, when complexedto an Env protein, to inhibit CD4 binding thereto, and/or upregulate CD4inducible epitopes on the resulting Env protein-CD4 mimetic complex),the Applicant discovered that the small molecules shared certainstructural similarities with one another.

That is to say, preferred small molecule CD4 mimetics of the inventiontypically although do not necessarily comprise a fused bicyclic ortricyclic core structure. Illustrative core structures include aromaticring systems, optionally containing one or more heteroatoms, such as anindole, a pyrrolopyridine, and a fluorenone. Preferably, a smallmolecule CD4 mimetic in accordance with the invention comprises one ofthe following core structures:

where in structure V, none or one of ring carbons 4, 5, 6, or 7 may bereplaced by a nitrogen. Structure V represents an indole core structure,while replacement of any one of ring carbons 4, 5, 6, or 7 results in apyrrolopyridine. Pyrrolopyridine core structures include:

Structure VI is a fluoren-9-one.

In certain instances, in each of core structures V and VI, one of thering atoms is covalently attached, via an optional linker, J, to asubstituted or unsubstituted piperidine or piperazine, which itself mayform part of a bicyclic ring structure, such as atetrahydro-isoquinolone, whose parent structure is shown below.

An illustrative bicyclic ring structure formed by a piperidine includesthe following illustrative substituted tetrahydro-isoquinolone, althoughany of a number of different substituents may be present on thetetrahydro-isoquinolone ring.

Any of a number of optional substituents may be present on the corestructures, that is to say, covalently attached to one or more ring atompositions. For example, suitable substituents include C₁-C₆ alkyl, C₃-C₆cycloalkyl, C₂-C₆ alkenyl, C₄-C₆ cycloalkenyl, C₂-C₆ alkynyl, halogen,—CN, phenyl, benzyl, nitro, C₁-C₆ alkoxy, C₃-C₆ cycloalkoxy, benzyloxy,—C(O)—C₁-C₆ alkyl, —C(O)—C₆H₅—, and, for example, —C(O)—C₆H₄—W, whereinW is an ortho, meta, or para-halogen

As stated previously, the small molecule CD4 mimetic may optionallycomprise a linker, J. Typically, the linker J ranges from about 1 toabout 50 atoms in length. More preferably, J ranges from about 1 toabout 40 atoms in length, or from about 1 to about 30 atoms in length.Representative linkers shown explicitly herein generally have a lengthof 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 19 or20 atoms.

Linkers of the invention include the following: —O—, —S—, —S—S—, —C(O)—,—O—C(O)—, —C(O)—O—, —C(O)—C(O)—, —C(O)—NH—, —NH—C(O)—NH—, —O—C(O)—NH—,—C(S)—, —CH₂—, —CH₂—CH₂—, —CH₂—CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂—, —O—CH₂—,—CH₂—O—, —O—CH₂—CH₂—, —CH₂—O—CH₂—, —CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—,—CH₂—O—CH₂—CH₂—, —CH₂—CH₂—O—CH₂—, —CH₂—CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—CH₂—,—CH₂—O—CH₂—CH₂—CH₂—, —CH₂—CH₂—O—CH₂—CH₂—, —CH₂—CH₂—CH₂—O—CH₂—,—CH₂—CH₂—CH₂—CH₂—O—, —CH(OH)CH₂—C(O)—NH—CH₂—, —C(O)—NH—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—C(O)—NH—, —C(O)—NH—CH₂—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—,—C(O)—NH—CH₂—CH₂—CH₂—CH₂—, —CH₂—C(O)—NH—CH₂—CH₂—CH₂—,—CH₂—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂—C(O)—NH—, —C(O)—O—CH₂—,CH₂—C(O)—O—CH₂—, —CH₂—CH₂—C(O)—O—CH₂—, —C(O)—O—CH₂—CH₂—, —NH—C(O)—CH₂—,—NH—C(O)-furanyl, —CH₂—NH—C(O)—CH₂—, —CH₂—CH₂—NH—C(O)—CH₂—,—NH—C(O)—CH₂—CH₂—, —CH₂—NH—C(O)—CH₂—CH₂—, —CH₂—CH₂—NH—C(O)—CH₂—CH₂—,—C(O)—NH—CH₂—, —C(O)—NH—CH₂—CH₂—, —O—C(O)—NH—CH₂—, —O—C(O)—NH—CH₂—CH₂—,—O—C(O)—NH—CH₂—CH₂—CH₂—, —NH—CH₂—, —NH—CH₂—CH₂—, —CH₂—NH—CH₂—,—CH₂—CH₂—NH—CH₂—, —C(O)—CH₂—, —C(O)—CH(CH₃)—, —C(O)—CH₂—CH₂—,—CH₂—C(O)—CH₂—, —CH₂—CH₂—C(O)—CH₂—, —CH₂—CH₂—C(O)—CH₂—CH₂—,—CH₂—CH₂—C(O)—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—,

—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—,

—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—CH₂—,—O—C(O)—NH—[CH₂]₀₋₆—(OCH₂CH₂)₀₋₂—, —C(O)—NH—(CH₂)₁₋₆—NH—C(O)—,—NH—C(O)—NH—(CH₂)₁₋₆—NH—C(O)—, —O—C(O)—CH₂—, —O—C(O)—CH₂—CH₂—, and—O—C(O)—CH₂—CH₂—CH₂—. Preferred linkers include, in one embodiment,—C(O)CH₂— —C(O)—, —C(O)—CHCH₃—, —C(O)—C(O)—, —CH(OH)CH₂—, —CH₂—,—N—C(O)-furanyl, in either orientation.

In yet another embodiment, a small molecule CD4 mimetic in accordancewith the invention is characterized by one of the following structures:

-   -   where in each of structures I, II and III:

W, X, Y, and Z are each independently selected from the group consistingof carbon and nitrogen, provided that no more than one of W, X, Y, or Zis nitrogen;

R₁, R′₁, R₂, R₃, R₄, R₅ are each independently selected from the groupconsisting of H, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, C₂-C₆ alkenyl, C₄-C₆cycloalkenyl, C₂-C₆ alkynyl, halogen, CN, phenyl, nitro, OC(O)R₁₅,C(O)R₁₅, C(O)OR₁₆, C(O)NR₁₇R₁₈, OR₁₉, SR₂₀, and NR₂₁R₂₂, each of saidalkyl and cycloalkyl being optionally substituted with one to threesubstituents independently selected from halogen, CN, NO₂, OC(O)R₁₅,C(O)R₁₅, C(O)OR₁₅, C(O)NR₁₇R₁₈, OR₁₉, SR₂₀, and NR₂₁R₂₂, with theproviso that R₂, R₃, R₄, or R₅ is absent when the ring atom to which itis attached is a double-bonded nitrogen, i.e., a trivalent nitrogen;

R₁₅, is independently selected from the group consisting of H, C₁-C₆alkyl, C₃-C₆ cycloalkyl, C₂-C₆ alkenyl, and C₄-C₆ cycloalkenyl;

R₁₆, R₁₉, and R₂₀ are each independently selected from the groupconsisting of H, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, C₂-C₆ alkenyl, C₄-C₆cycloalkenyl, C₃-C₆ alkynyl, phenyl, and benzyl, wherein each of saidalkyl, cycloalkyl, phenyl, and benzyl are optionally substituted withone, two, or three substituents selected from the group consisting ofhalogen, NR₂₁R₂₂, OH, CN, or NO₂; provided the carbon atoms thatcomprise the carbon-carbon triple bond of said C₃-C₆ alkynyl are not thepoint of attachment to the oxygen or sulfur to which R₁₆, R₁₉, or R₂₀ isattached;

R₁₇ and R₁₈ are each independently selected from the group consisting ofH, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, C₃-C₆ alkenyl, C₄-C₆ cycloalkenyl, andC₃-C₆ alkynyl, wherein each of said alkyl and cycloalkyl are optionallysubstituted with one, two, or three substituents selected from the groupconsisting of halogen, NR₂₁R₂₂, OH, CN, or NO₂; provided the carbonatoms that comprise the carbon-carbon double bond of said C₃-C₆ alkenylor the carbon-carbon triple bond of said C₃-C₆ alkynyl are not the pointof attachment to the nitrogen to which R₁₇ and R₁₈ is attached;

R₂₁ and R₂₂ are each independently selected from the group consisting ofH, OH, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, C₃-C₆ alkenyl, C₅-C₆ cycloalkenyl,C₃-C₆ alkynyl, phenyl, and C(O)R₂₃; provided the carbon atoms thatcomprise the carbon-carbon double bond of said C₃-C₆ alkenyl, C₄-C₆cycloalkenyl, or the carbon-carbon triple bond of said C₃-C₆ alkynyl arenot the point of attachment to the nitrogen to which R₂₁ and R₂₂ isattached;

R₂₃ is selected from the group consisting of H, C₁-C₆ alkyl, C₃-C₆cycloalkyl, C₂-C₆ alkenyl, C₄-C₆ cycloalkenyl, and C₂-C₆ alkynyl;

R₆ is (CH₂)_(n)Q, wherein n is 0-6;

Q is selected from:

-   -   (1) H, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, C₂-C₆ alkenyl, C₄-C₆        cycloalkenyl, C₃-C₆ alkynyl, halogen, CN, Ar, NO₂, C(O)R₂₄,        C(O)OR₂₅, C(O)NR₂₆R₂₇, COOAr, TR₆, NR₂₁R₂₂, NC(O)NR₂₁R₂₂,        OC(O)R₁₆, (N(R₂₁)₂)C═N-T-R₂₂, C(O)Ar, or S(O)_(m)R₂₁;    -   (2) a 4-7 membered heterocyclic ring, optionally substituted        with R₁₆, which may contain 1-3 heteroatoms selected from the        group consisting of O, S, SO, SO₂, N, and NR₂₄;

T is S or O;

m is 0-2;

R₂₄ is selected from the group consisting of H, C₁-C₆ alkyl, C₃-C₆cycloalkyl, C₃-C₆ alkenyl, C₄-C₆ cycloalkenyl, and C₃-C₆ alkynyl;

R₂₅ is selected from the group consisting of C₁-C₆ alkyl, C₃-C₆cycloalkyl, C₂-C₆ alkenyl, C₄-C₆ cycloalkenyl, and C₃-C₆ alkynyl;provided the carbon atoms that comprise the carbon-carbon triple bond ofsaid C₃-C₆ alkynyl are not the point of attachment to the oxygen towhich R₂₅ is attached;

R₂₆ and R₂₇ are each independently selected from the group consisting ofH, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, C₃-C₆ alkenyl, C₅-C₆ cycloalkenyl, andC₃-C₆ alkynyl; provided the carbon atoms that comprise the carbon-carbondouble bond of said C₃-C₆ alkenyl, C₅-C₆ cycloalkenyl, or thecarbon-carbon triple bond of said C₃-C₆ alkynyl are not the point ofattachment to the nitrogen to which R₂₆ and R₂₇ are attached;

R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃, and R₁₄ are each independently selectedfrom the group consisting of H, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, C₂-C₆alkenyl, C₄-C₆ cycloalkenyl, C₂-C₆ alkynyl, CR₂₈R₂₉OR₃₀, C(O)R₃₁,CR₃₂(OR₃₃)OR₃₄, CR₃₅NR₃₆R₃₇, C(O)OR₃₈, C(O)NR₃₉R₄₀, CR₄₁R₄₂F, CR₄₃F₂,and CF₃, each of said alkyl and cycloalkyl being optionally substitutedwith one to three substituents independently selected from halogen, CN,NO₂, OC(O)R₁₅, C(O)R₁₅, C(O)OR₁₅, C(O)NR₁₇R₁₈, OR₁₉, SR₂₀, and NR₂₁R₂₂,or R₈ and R₉ may be absent such that the carbon ring atoms to which eachis independently attached together form a double bond.

R₂₈, R₂₉, R₃₀, R₃₁, R₃₂, R₃₅, R₄₁, R₄₂ and R₄₃ are each independentlyselected from the group consisting of H, C₁-C₆ alkyl, C₃-C₆ cycloalkyl,C₂-C₆ alkenyl, C₄-C₆ cycloalkenyl, C₂-C₆ alkynyl, and C(O)R₄₄;

R₃₃, R₃₄ and R₃₈ are each independently selected from the groupconsisting of H, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, C₃-C₆ alkenyl, C₄-C₆cycloalkenyl, and C₃-C₆ alkynyl; provided the carbon atoms that comprisethe carbon-carbon triple bond of said C₃-C₆ alkynyl are not the point ofattachment to the oxygen to which R₃₄ and R₃₈ are attached;

R₃₆ and R₃₇ are each independently selected from the group consisting ofH, C₁-C₆ alkyl, C₁-C₆ cycloalkyl, C₃-C₆ alkenyl, C₄-C₆ cycloalkenyl, andC₃-C₆ alkynyl; provided the carbon atoms that comprise the carbon-carbontriple bond of said C₃-C₆ alkynyl are not the point of attachment to thenitrogen to which R₃₆ and R₃₇ are attached;

R₃₉ and R₄₀ are each independently selected from the group consisting ofH, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, C₂-C₆ alkenyl, C₄-C₆ cycloalkenyl, andC₃-C₆ alkynyl; provided the carbon atoms that comprise the carbon-carbontriple bond of said C₃-C₆ alkynyl are not the point of attachment to thenitrogen to which R₃₉ and R₄₀ are attached; R₄₄ is selected from thegroup consisting of H, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, C₂-C₆ alkenyl,C₄-C₆ cycloalkenyl, and C₂-C₆ alkynyl;

J is —(CO)—, —(CO)CH₂—, —(CO)(CO)—, or —(CO)CHR₉—;

U is nitrogen or CH;

L is —(CO)— or —CH₂—;

R_(A), R_(B), and R_(C) are independently selected from the groupconsisting of Ar, H, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, C₂-C₆ alkenyl, C₄-C₆cycloalkenyl, C₂-C₆ alkynyl, halogen, CN, nitro, OC(O)R₁₅, C(O)R₁₅,C(O)OR₁₆, C(O)NR₁₇R₁₈, OR₁₉, SR₂₀ and NR₂₁R₂₂, each of said alkyl andcycloalkyl being optionally substituted with one to three substituentsindependently selected from halogen, CN, NO₂, OC(O)R₁₅, C(O)R₁₅,C(O)OR₁₅, C(O)NR₁₇R₁₈, OR₁₉, SR₂₀, and NR₂₁R₂₂;

Ar is a 5-12 membered aromatic ring optionally containing one to fiveheteroatoms independently selected from the group consisting of O, S, N,or NR₁₆ and optionally substituted with one to six substitutentsindependently selected from the group consisting of R₄₅, R₄₆, R₄₇, R₄₈,or R₄₉;

R₄₅ R₄₆, R₄₇, R₄₈, and R₄₉ are each independently H, C₁-C₆ alkyl, C₃-C₆cycloalkyl, C₂-C₆ alkenyl, C₃-C₆ cycloalkenyl, C₂-C₆ alkynyl, halogen,CN, nitro, C(O)R₄₅, COOR₄₆, hydroxyl, hydroxymethyl, trifluoromethyl,trifluoromethoxy, O-benzyl, O-phenyl, OC(O)C₁-C₆ alkyl, SC(O)C₁-C₆alkyl, S(O)_(m)C₁-C₆ alkyl, S(O)₂NR₂₁R₂₂, O—V, CH₂ (CH₂)_(p)—V,O(CH₂)—V, (CH₂)_(p)O—V, CH═CH—V, or TR₄₇, each of said alkyl andcycloalkyl being optionally substituted with one to three substitutentsindependently selected from the group consisting of, NR₂₁NR₂₂, OH, CN,or NO₂;

V is 4-methoxyphenyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, pyrazyl,quinolyl, 3,5-dimethylisoxazoyl, isoxazoyl, 2-methylthiazoyl, thiazoyl,2-thienyl, 3-thienyl, or pyrimidyl;

p is 1-2;

R₄₅ and R₄₆ are each independently H, C₁-C₆ alkyl, or C₃-C₆ cycloalkyl;

R₄₇ is H, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, C₂-C₆ alkenyl, C₃-C₆cycloalkenyl, C₂-C₆ alkynyl, C(O)R₄₈ or C(O)OR₄₉, each of said alkyl andcycloalkyl being optionally substituted with one to three substitutentsindependently selected from the group consisting of, NR₂₁NR₂₂, OH, CN,or NO₂; and

R₄₈, R₄₉ are each independently H, C₁-C₆ alkyl, or C₃-C₆ cycloalkyl,each of said alkyl and cycloalkyl being optionally substituted with oneto three substitutents independently selected from the group consistingof, NR₂₁NR₂₂, OH, CN, or NO₂.

Small molecule CD4 mimetics in accordance with the invention exclude thefollowing:

Additional illustrative small molecule CD4 mimetics (compounds I-30) inaccordance with the invention and their corresponding structures areprovided in Tables 1-4 herein. For instance, preferred small moleculeCD4 mimetics include1-[4-(1H-Indole-6-carbonyl)-piperazin-1-yl]-2,2-diphenyl-ethanone (11);[4-(1H-Indole-6-carbonyl)-piperazin-1-yl]-(3,4,5-trimethoxy-phenyl)-methanone(8);[4-(1H-Indol-3-yl)-piperidin-1-yl]-(3,4,5-trimethoxy-phenyl)-methanone(12);(5-Benzyloxy-3-methyl-1H-indol-2-yl)-(4-methyl-piperazin-1-yl)-methanone(13); (5-Benzyloxy-3-methyl-1H-indol-2-yl)-morpholin-4-yl-methanone(14);2-{2-[2-(2-Methyl-1H-indol-3-yl)-2-oxo-ethyl]-1,2,3,4-tetrahydro-isoquinolin-1-ylmethyl}-isoindole-1,3-dione(15);1-[2-(2-Methyl-propenyl)-1H-indol-3-yl]-2-morpholin-4-yl-ethane-1,2-dione(16);(5-Methoxy-1,2-dimethyl-1H-indol-3-yl)-(4-methyl-piperazin-1-yl)-methanone(17); and2-[1-(4-Chloro-benzoyl)-5-methoxy-2-methyl-1H-indol-3-yl]-1-(4-phenyl-piperazin-1-yl)-ethanone(18), 2-(4-Benzyl-piperazin-1-yl)-1-(5-methoxy-1H-indol-3-yl)-ethanone(2);2-{2-[2-(1H-Indol-3-yl)-2-oxo-ethyl]-1,2,3,4-tetrahydro-isoquinolin-1-ylmethyl}-isoindole-1,3-dione(20);1-[2-(5-Methoxy-1,2-dimethyl-1H-indol-3-yl)-2-oxo-ethyl]-piperidine-4-carboxylicacid amide (21);4-[2-Hydroxy-2-(5-methoxy-1H-indol-3-yl)-ethyl]-piperazine-1-carboxylicacid ethyl ester (22);1-[2-(5-Methoxy-1H-indol-3-yl)-2-oxo-ethyl]-piperidine-4-carboxylic acidethyl ester (23), 1-(1H-Indol-3-yl)-2-piperidin-1-yl-ethane-1,2-dione(19);[4-(1H-Indol-3-ylmethyl)-piperazin-1-yl]-(3,4,5-trimethoxy-phenyl)-methanone(20); 1-(1H-Indol-3-yl)-2-morpholin-4-yl-ethane-1,2-dione (21);(5-Benzyloxy-3-methyl-1H-indol-2-yl)-piperidin-1-yl-methanone (22);Furan-2-carboxylic acid(1-benzyl-piperidin-4-yl)-(2,3-dimethyl-1H-indol-7-yl)-amide (23);(5-Benzyloxy-3-methyl-1H-indol-2-yl)-(4-phenyl-piperazin-1-yl)-methanone(24);[4-(1H-Indol-3-ylmethyl)-piperazin-1-yl]-(2-methoxy-phenyl)-methanone(25);2-[4-(2-Methoxy-phenyl)-piperazin-1-yl]-1-(2-methyl-1H-indol-3-yl)-ethanone(26);2-{2-[2-Hydroxy-2-(5-methoxy-1H-indol-3-yl)-ethyl]-1,2,3,4-tetrahydro-isoquinolin-1-ylmethyl}-isoindole-1,3-dione(27);2-[4-(2-Hydroxy-ethyl)-piperazin-1-yl]-1-(5-methoxy-1H-indol-3-yl)-ethanone(28);2-{2-[2-(5-Methoxy-1H-indol-3-yl)-2-oxo-ethyl]-1,2,3,4-tetrahydro-isoquinolin-1-ylmethyl}-isoindole-1,3-dione(29);{4-[2-(5-Methoxy-1H-indol-3-yl)-2-oxo-ethyl]-piperazin-1-yl}-acetic acidmethyl ester (30);1-[2-(5-methoxy-1H-indol-3-yl)-2-oxoethyl]piperidine-4-carboxamide (1);2-(4-benzylpiperazin-1-yl)-1-(5-methoxy-1H-indol-3-yl)ethanone (2);{1-[(4,6-dimethyl-1H-indol-2-yl)carbonyl]piperidin-4-yl}(4-fluorophenyl)methanone(3);1-[2-(5-methoxy-1,2-dimethyl-1H-indol-3-yl)-1-methyl-2-oxoethyl]piperidine-4-carboxamide(4); ethyl1-[(4,6-dimethyl-1H-indol-2-yl)carbonyl]piperidine-4-carboxylate (5);6-{[4-(2,6-difluorobenzoyl)piperazin-1-yl]carbonyl}-1H-indole (6);2-{[4-(1H-indol-6-ylcarbonyl)piperazin-1-yl]carbonyl}phenyl acetate (7);6-{[4-(3,4,5-trimethoxybenzoyl)piperazin-1-yl]carbonyl}-1H-indole (8);1-[2-(5-methoxy-1,2-dimethyl-1H-indol-3-yl)-2-oxoethyl]piperidine-4-carboxamide(9); and 4-{[4-(2-furoyl)piperazin-1-yl]carbonyl}-9H-fluoren-9-one (10).

Peptoids

Peptoids are an easily synthesized class of peptidomimetic oligomer thatare highly diverse in structure and are stable to enzymatic and chemicaldegradation (Miller, S. M. et al. Proteolytic Studies of Homologouspeptide and N-Substituted Glycine Peptoid Oligomers. Bioorg. Med. Chem.Lett. (1994), 4, 2657-2662).

Hundreds of different side chains can be incorporated into the polymer,including polar, reactive and even heterocyclic functionalities[Zuckermann, R. N., Kerr, J. M., Kent, S. B. H. & Moos, W. H. EfficientMethod for the Preparation of Peptoids [Oligo(N-substituted glycines)]by Submonomer Solid Phase Synthesis. J. Am. Chem. Soc. (1992), 114,10646-10647; Figliozzi, G. M., Goldsmith, R., Ng, S., Banville, S. C. &Zuckermann, R. N. Synthesis of N-(substituted)glycine Peptoid Libraries.Methods Enzymol. (1996), 267, 437-447; Burkoth, T. S., Fafarman, A. T.,Charych, D. H., Connolly, M. D. & Zuckermann, R. N. Incorporation ofUnprotected Heterocyclic Side Chains into Peptoid Oligomers viaSolid-Phase Submonomer Synthesis. J. Am. Chem. Soc. (2003), 125,8841-8845; Uno, T., Beausoleil, E., Goldsmith, R. A., Levine, B. H. &Zuckermann, R. N. New Submonomers for Poly N-Substituted Glycines(Peptoids). Tetrahedron Lett. (1999), 40, 1475-1478]. The ability toefficiently incorporate a wide variety of structural features allowsrapid synthesis of biomimetic oligomers with far more chemical diversitythan natural peptides.

The present invention provides peptoid CD4 mimetics. Thus in someembodiments, the present invention provides peptoid mimics of key CD4binding residues such as Phe43 and Arg59. In other embodiments, thepeptoids further mimic the CD4 beta-turn motif by incorporating abeta-turn inducing amino acid such asFmoc-3-amino-1-carboxymethylcaprolactam.

In one embodiment, the invention provides peptoids having at leasttwelve monomer units. In other embodiments, the invention providespeptoids having at least ten, nine, eight, seven, six, five, four, orthree monomer units.

In some embodiments, the peptoids provided by the present invention arelinked to small molecules to form a peptoid small molecule conjugate. Insome particular embodiments, the small molecules are modified with apeptoid trimer. Methods to conjugate small molecules to combinatoriallibraries of peptoid oligomers are known (Horn, T., Lee, B.-C., Dill, K.A. & Zuckermann, R. N. Incorporation of Chemoselective Functionalitiesinto Peptoids via Solid-Phase Submonomer Synthesis. Bioconj. Chem. 15,428-435 (2004). The method involves the solid-phase synthesis of anoligomer library, where each compound is functionalized with achemoselective ligation group, facilitating the attachment of the smallmolecule to a specific position of the oligomer. Alternatively, aminogroups are introduced into tolerant positions of the small molecule andthe amino-small molecule is used as a peptoid submonomer to beincorporated at any position of the oligomer. These positions are thenmodified with either amino or carboxyl groups to facilitate theirattachment to, or incorporation in a combinatorial oligomer library.

Crosslinked Peptoids, Small Molecules, and Peptoid Small MoleculeConjugates

In some embodiments, the invention provides peptoids, small molecules,and peptoid small molecule conjugates that are crosslinked to an Envpolypeptide. In some particular embodiments, the invention providespeptoids, small molecules, and peptoid small molecule conjugates havinga spacer and crosslinking moiety.

The spacer or linker varies in length and, in some embodiments, isconstructed from a combination of various aminoalkyl acids and di-acidsto span distances from 5-50 angstroms. Suitable spacers typically havean atom length of from about 3 to about 50 atoms. A cross linking moietycomprising a terminal reactive group (e.g. benzophenone) is placed atthe end of the linker. After contacting the Env polypeptide with thepeptoid, small molecule, or peptoid small molecule conjugate, both theEnv polypeptide and the compound are then reacted to form a covalentlybound Env polypeptide/compound complex.

Env Polypeptides

The Env polypeptide portion of the complexes described herein can bederived from an envelope protein, preferably from HIV Env. As notedabove, the envelope protein of HIV-1 is a glycoprotein of about 160 kd(gp160). During virus infection of the host cell, gp160 is cleaved byhost cell proteases to form gp120 and the integral membrane protein,gp41. The gp41 portion is anchored in (and spans) the membrane bilayerof virion, while the gp120 segment protrudes into the surroundingenvironment. As there is no covalent attachment between gp120 and gp41,free gp120 is released from the surface of virions and infected cells.Env polypeptides may also include gp140 polypeptides.

In certain embodiments, the Env polypeptide component of the complex isa monomer or a dimer. In preferred embodiments, the Env polypeptidecomponent is an oligomeric Env polypeptide (e.g., a trimer). The primarysequence of the Env polypeptide precursor of HIV-1_(SF)2 (hereinafter“SF2”) strain is known. See, e.g., FIG. 1 of International PublicationWO 04/037847. Env polypeptides contain multiple N-linked glycosylationsites, for example the HIV strain SF2 contains approximately 24 N-linkedglycosylation sites. As suggested by their name, the hypervariabledomains contain extensive amino acid substitutions, insertions anddeletions as between strains. Despite this variation, most, if not all,Env polypeptide sequences preserve the virus's ability to bind to theviral receptor CD4. Further, alignment of the amino acid sequences ofEnv polypeptide of any HIV variant can be determined relative to othervariants, such as HXB-2, as described for example, in WO 00/39303. Inother embodiments, the Env polypeptide comprises an oligomeric form ofEnv, for example oligomeric gp140 (o-gp140).

The Env polypeptide component of the Env-CD4 complex can be derived anyknown HIV isolates, as well as newly identified isolates, and subtypesof these isolates. Descriptions of structural features can be givenherein with reference to SF2 or HXB-2. One of ordinary skill in the artin view of the teachings of the present disclosure and the art candetermine corresponding regions in other HIV variants (e.g., isolatesHIV_(IIIb), HIV-1_(SF162), HIV-1_(SF170), HIV_(LAV), HIV_(LAI),HIV_(MN), HIV-1_(CM235), HIV-1_(US4), other HIV-1 strains from diversesubtypes (e.g., subtypes, A through G, and O), HIV-2 strains and diversesubtypes (e.g., HIV-2_(UC1) and HIV-2_(UC2)), and simianimmunodeficiency virus (SIV). (See, e.g., Virology, 3rd Edition (W. K.Joklik ed. 1988); Fundamental Virology, 2nd Edition (B. N. Fields and D.M. Knipe, eds. 1991); Virology, 3rd Edition (Fields, B N, D M Knipe, P MHowley, Editors, 1996, Lippincott-Raven, Philadelphia, Pa.; for adescription of these and other related viruses), using for example,sequence comparison programs (e.g., BLAST and others described herein)or identification and alignment of structural features (e.g., a programsuch as the “ALB” program described herein that can identify O-sheetregions). The actual amino acid sequences of the modified Envpolypeptides can be based on any HIV variant.

The Env polypeptides described herein may include additionalmodifications to the native sequence, such as additional internaldeletions, additions and substitutions. These modifications may bedeliberate, as through site-directed mutagenesis, or may be accidental,such as through naturally occurring mutational events. Thus, forexample, if the Env polypeptide is to be used in vaccine compositions,the modifications must be such that immunological activity (i.e., theability to elicit an antibody response to the polypeptide) is not lost.Similarly, if the polypeptides are to be used for diagnostic purposes,such capability must be retained. The Env polypeptides described hereincan be monomeric or oligomeric.

The Env polypeptides of the present invention can be produced in anynumber of ways all of which are well known in the art, including, forexample, peptide synthesis techniques, recombinant production and thelike. See, e.g., Srivastava et al. (2003) J. Virol. 77(20):1 1244-11259describing production of oligomeric Env proteins.

Env-CD4 Complexes

The present invention also encompasses complexes of CD4 mimetics and Envpolypeptides that are effective to change the conformation of the Envpolypeptide and expose epitopes that elicit production of antibodies,particularly neutralizing antibodies. The Env-CD4 mimetic complexes maybe formed in vivo or, alternatively, complexes may be formed in vitro orex vivo, for example in order to generate antibody production. Theantibodies may then be used in pharmaceutical compositions foradministration to a subject.

Env and CD4 mimetics as described herein can be complexed in a varietyof ways. In certain embodiments, Env and CD4 proteins are complexed in areaction mixture, for example using one or more cross-linking agents,such as formaldehyde, glutyraldehyde and the like. In other embodiments,a CD4 mimetic is linked to the envelope by a specific covalent bondwhich will not perturb the envelope exposed antigenic surface, yetexposing the cryptic conserved epitopes that are normally notaccessible, for example so that an antibody response can be mounted. Instill further embodiments, a fixative such as formalin is used tocomplex Env and CD4 proteins. CD4 mimetics are complexed or linked withEnv to maintain the exposure of CD4-inducible epitopes in the Envprotein for immunization purposes to target the functional epitopes forvaccine applications.

A pH near physiological pH is most preferred since it is believed tomore accurately mimic binding of CD4 to Env in vivo. The most favorablepH for promoting complexing can be determined by one skilled in the art,and will depend upon the particular protein to be modified. Suitablebuffers include sodium phosphate, sodium acetate, sodium carbonate, andphosphate buffered saline (PBS). Typically, the CD4 mimetic is added tothe protein-containing solution at an equimolar amount or at a molarexcess relative to target protein, for instance molar ratio of about1:1, 1.5:1, 2:1, 3:1, 4:1, 5:1, 6:1, 8:1, or 10:1.

The complexing reactions are typically carried out at temperatures at ornear about physiological temperatures (37° C.), although temperaturesmay range from about −15° C. to about 45° C., more preferably from about25° C. to 40° C., for approximately one to twenty four hours. The exactreaction time may vary. Binding of CD4 mimetics to Env can be determinedusing a variety of techniques, for example, HPLC, ELISA and Biacore'ssurface plasmon resonance (SPR) systems (Biacore Inc, Piscataway, N.J.).See, also, FIGs 1 and 2.

In addition, suitable CD4 mimetic-Env complexes may be produced in vivo,by administering one or more CD4 mimetics as described herein to asubject under conditions such that the CD4 mimetic binds to Envpolypeptides in the subject. Administration of CD4 mimetics to thesubject can be readily accomplished and is described below.

Antibodies

Antibodies, both monoclonal and polyclonal, which are directed againstepitopes on Env-CD4 mimetic complexes (and cryptic epitopes exposed bybinding of CD4 mimetics to Env) are particularly useful in diagnosis andtherapeutic applications, for example, those antibodies which areneutralizing are useful in passive immunotherapy. Monoclonal antibodies,in particular, may be used to raise anti-idiotype antibodies.

Anti-idiotype antibodies are immunoglobulins that carry an “internalimage” of the antigen of the infectious agent against which protectionis desired. Techniques for raising anti-idiotype antibodies are known inthe art. See, e.g., Grzych (1985), Nature 316:74; MacNamara et al.(1984), Science 226:1325, Uytdehaag et al (1985), J. Immunol. 134:1225.These anti-idiotype antibodies may also be useful for treatment and/ordiagnosis of HIV.

An immunoassay for viral antigen may use, for example, a monoclonalantibody directed towards a viral epitope, a combination of monoclonalantibodies directed towards epitopes of one viral polypeptide,monoclonal antibodies directed towards epitopes of different viralpolypeptides, polyclonal antibodies directed towards the same viralantigen, polyclonal antibodies directed towards different viral antigensor a combination of monoclonal and polyclonal antibodies.

Immunoassay protocols may be based, for example, upon competition, ordirect reaction, or sandwich type assays. Protocols may also, forexample, use solid supports, or may be by immunoprecipitation. Mostassays involve the use of labeled antibody or polypeptide. The labelsmay be, for example, fluorescent, chemiluminescent, radioactive, or dyemolecules. Assays which amplify the signals from the probe are alsoknown. Examples of which are assays which utilize biotin and avidin, andenzyme-labeled and mediated immunoassays, such as ELISA assays.

An enzyme-linked immunosorbent assay (ELISA) can be used to measureeither antigen or antibody concentrations. This method depends uponconjugation of an enzyme to either an antigen or an antibody, and usesthe bound enzyme activity as a quantitative label. To measure antibody,the known antigen is fixed to a solid phase (e.g., a microplate orplastic cup), incubated with test serum dilutions, washed, incubatedwith anti-immunoglobulin labeled with an enzyme, and washed again.Enzymes suitable for labeling are known in the art, and include, forexample, horseradish peroxidase. Enzyme activity bound to the solidphase is measured by adding the specific substrate, and determiningproduct formation or substrate utilization colorimetrically. The enzymeactivity bound is a direct function of the amount of antibody bound.

To measure antigen, a known specific antibody is fixed to the solidphase, the test material containing antigen is added, after anincubation the solid phase is washed, and a second enzyme-labeledantibody is added. After washing, substrate is added, and enzymeactivity is estimated colorimetrically, and related to antigenconcentration.

Polyclonal antibodies can be produced by administering the fusionprotein to a mammal, such as a mouse, a rabbit, a goat, or a horse.Serum from the immunized animal is collected and the antibodies arepurified from the plasma by, for example, precipitation with ammoniumsulfate, followed by chromatography, preferably affinity chromatography.Techniques for producing and processing polyclonal antisera are known inthe art.

Monoclonal antibodies directed against epitopes exposed by binding ofCD4 to Env can also be produced. Normal B cells from a mammal, such as amouse, immunized with, e.g., an Env-CD4 complex as described herein canbe fused with, for example, HAT-sensitive mouse myeloma cells to producehybridomas. Hybridomas producing antibodies specific for epitopesexposed when CD4 miniproteins bind to Env can be identified using RIA orELISA and isolated by cloning in semi-solid agar or by limitingdilution. Clones producing the desired specific antibodies are isolatedby another round of screening.

Antibodies, monoclonal and/or polyclonal, which are directed againstepitopes, are particularly useful for detecting the presence of antigensin a sample, such as a serum sample from an HIV-infected human. Animmunoassay for an HIV antigen may utilize one antibody or severalantibodies. An immunoassay for an HIV antigen may use, for example, amonoclonal antibody directed towards an HIV epitope, a combination ofmonoclonal antibodies directed towards epitopes of one Env or Env-CD4polypeptide, monoclonal antibodies directed towards epitopes ofdifferent polypeptides, polyclonal antibodies directed towards the sameHIV antigen, polyclonal antibodies directed towards different HIVantigens, or a combination of monoclonal and polyclonal antibodies.Immunoassay protocols may be based, for example, upon competition,direct reaction, or sandwich type assays using, for example, labeledantibody. The labels may be, for example, fluorescent, chemiluminescent,or radioactive.

The polyclonal or monoclonal antibodies may further be used to isolateEnv or CD4 complexed-Env by immunoaffinity columns. The antibodies canbe affixed to a solid support by, for example, adsorption or by covalentlinkage so that the antibodies retain their immunoselective activity.Optionally, spacer groups may be included so that the antigen bindingsite of the antibody remains accessible. The immobilized antibodies canthen be used to bind the target from a biological sample, such as bloodor plasma. The bound proteins or complexes are recovered from the columnmatrix by, for example, a change in pH.

Diagnostic, Vaccine and Therapeutic Applications

The CD4 mimetics, Env-CD4 mimetic complexes and antibodies thereto canbe used for a number of diagnostic and therapeutic purposes. Forexample, as noted above, the antibodies generated against the same, canbe used in a variety of assays, to determine the presence of reactiveantibodies/and or Env proteins in a biological sample to aid in thediagnosis of HIV infection or disease status or as measure of responseto immunization.

As noted above, the presence of antibodies reactive with the Env (e.g.,gp120) polypeptides and, conversely, antigens reactive with antibodiesgenerated thereto, can be detected using standard electrophoretic andimmunodiagnostic techniques, including immunoassays such as competition,direct reaction, or sandwich type assays. Such assays include, but arenot limited to, western blots; agglutination tests; enzyme-labeled andmediated immunoassays, such as ELISAs; biotin/avidin type assays;radioimmunoassays; immunoelectrophoresis; immunoprecipitation, etc. Thereactions generally include revealing labels such as fluorescent,chemiluminescent, radioactive, or enzymatic labels or dye molecules, orother methods for detecting the formation of a complex between theantigen and the antibody or antibodies reacted therewith.

Solid supports can be used in the assays such as nitrocellulose, inmembrane or microtiter well form; polyvinylchloride, in sheets ormicrotiter wells; polystyrene latex, in beads or microtiter plates;polyvinylidine fluoride; diazotized paper; nylon membranes; activatedbeads, and the like.

Typically, the solid support is first reacted with the biological sample(or the gp120 proteins), washed and then the antibodies, (or a samplesuspected of containing antibodies), applied. After washing to removeany non-bound ligand, a secondary binder moiety is added under suitablebinding conditions, such that the secondary binder is capable ofassociating selectively with the bound ligand. The presence of thesecondary binder can then be detected using techniques well known in theart. Typically, the secondary binder will comprise an antibody directedagainst the antibody ligands. A number of anti-human immunoglobulin (Ig)molecules are known in the art (e.g., commercially available goatanti-human Ig or rabbit anti-human Ig). Ig molecules for use herein willpreferably be of the IgG or IgA type, however, IgM may also beappropriate in some instances. The Ig molecules can be readilyconjugated to a detectable enzyme label, such as horseradish peroxidase,glucose oxidase, Beta-galactosidase, alkaline phosphatase and urease,among others, using methods known to those of skill in the art. Anappropriate enzyme substrate is then used to generate a detectablesignal.

In another embodiment, a “two antibody sandwich” assay can be used todetect the proteins of the present invention. In this technique, thesolid support is reacted first with one or more of the antibodiesdirected against Env-CD4 mimetic complexes, washed and then exposed tothe test sample. Antibodies are again added and the reaction visualizedusing either a direct color reaction or using a labeled second antibody,such as an anti-immunoglobulin labeled with horseradish peroxidase,alkaline phosphatase or urease.

Assays can also be conducted in solution, such that the viral proteinsand antibodies thereto form complexes under precipitating conditions.The precipitated complexes can then be separated from the test sample,for example, by centrifugation. The reaction mixture can be analyzed todetermine the presence or absence of antibody-antigen complexes usingany of a number of standard methods, such as those immunodiagnosticmethods described above.

The Env-CD4 complexes, produced as described above, or antibodies to thecomplexes, can be provided in kits, with suitable instructions and othernecessary reagents, in order to conduct immunoassays as described above.The kit can also contain, depending on the particular immunoassay used,suitable labels and other packaged reagents and materials (i.e. washbuffers and the like). Standard immunoassays, such as those describedabove, can be conducted using these kits.

The CD4 mimetics, Env-CD4 mimetic complexes and antibodies thereto canalso be used in vaccine compositions, individually or in combination, ine.g., prophylactic (i.e., to prevent infection) or therapeutic (to treatHIV following infection) vaccines. The vaccines can comprise mixtures ofone or more of the modified Env proteins (or nucleotide sequencesencoding the proteins), such as Env (e.g., gp120) proteins derived frommore than one viral isolate. The vaccine may also be administered inconjunction with other antigens and immunoregulatory agents, forexample, immunoglobulins, cytokines, lymphokines, and chemokines,including but not limited to IL-2, modified IL-2 (cys125-ser125),GM-CSF, IL-12, -interferon, IP-10, MIP1 and RANTES. The vaccines may beadministered as polypeptides or, alternatively, as naked nucleic acidvaccines (e.g., DNA), using viral vectors (e.g., retroviral vectors,alphaviral vectors, adenoviral vectors, adeno-associated viral vectors)or non-viral vectors (e.g., liposomes, particles coated with nucleicacid or protein, including viral replicon particles). The vaccines mayalso comprise a mixture of protein and nucleic acid, which in turn maybe delivered using the same or different vehicles. The vaccine may begiven more than once (e.g., a “prime” administration followed by one ormore “boosts”) to achieve the desired effects. The same composition canbe administered as the prime and as the one or more boosts. In anotherembodiment, different compositions can be used for priming and boosting.

A number of viral based systems have been developed for delivery andadministration of nucleic acid molecules into mammalian cells. Forexample, retroviruses provide a convenient platform for nucleic aciddelivery systems. Selected sequences can be inserted into a vector andpackaged in retroviral particles using techniques known in the art. Therecombinant virus can then be isolated and delivered to cells of thesubject either in vivo or ex vivo. A number of retroviral systems havebeen described (U.S. Pat. No. 5,219,740; Miller and Rosman, (1989)BioTech. 7:980-990; Miller, A. D., (1990) Hum. Gene Ther. 1:5-14;Scarpa, et al., (1991) Virol. 180:849-852; Burns, et al. (1993) Proc.Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993)Curr. Opin. Genet. Develop. 3:102-109.

A number of adenovirus vectors have also been described. Unlikeretroviruses which integrate into the host genome, adenoviruses persistextrachromosomally thus minimizing the risks associated with insertionalmutagenesis (Haj-Ahmad and Graham, (1986) J. Virol. 57:267-274; Bett, etal., (1993) J. Virol. 67:5911-5921; Mittereder, et al., (1994) Hum. GeneTher. 5:717-729; Seth, et al., (1994) 1 Virol. 68:933-940; Barr, et al.,(1994) Gene Therapy 1:51-58; Berkner, K. L., (1988) BioTech. 6:616-629;and Rich, et al., (1993) Hum. Gene Ther. 4:461-476).

Additionally, various adeno-associated virus (AAV) vector systems havebeen developed for nucleic acid delivery and administration. AAV vectorscan be readily constructed using techniques well known in the art. See,e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; WO 92/01070; WO 93/03769;Lebkowski, et al. (1988) Mol. Cell. Biol. 8:3988-3996; Vincent, et al.,(1990) Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter, B. J.,(1992) Curr. Opin. Biotech. 3:533-539; Muzyczka, N., (1992) Curr. Top.Microbiol. Immunol. 158:97-129; Kotin, R. M., (1994) Hum. Gene Ther.5:793-801; Shelling and Smith, (1994) Gene Ther. 1:165-169; and Zhou, etal., (1994) J. Exp. Med. 179:1867-1875.

Another vector system useful for delivering polynucleotides is theenterically administered recombinant poxvirus vaccines described bySmall, Jr., P. A., et al. (U.S. Pat. No. 5,676,950).

Additional viral vectors which will find use for delivery andadministration of nucleic acid molecules include those derived from thepox family of viruses, including vaccinia virus and avian poxvirus. Inanother embodiment, avipoxviruses, such as the fowlpox and canarypoxviruses, can also be used for delivery of nucleic acid molecules.Recombinant avipox viruses, expressing immunogens from mammalianpathogens, are known to confer protective immunity when administered tonon-avian species. The use of an avipox vector is particularly desirablein human and other mammalian species since members of the avipox genuscan only productively replicate in susceptible avian species andtherefore are not infective in mammalian cells. Methods for producingrecombinant avipoxviruses are known in the art and employ geneticrecombination, as described above with respect to the production ofvaccinia viruses. See, e.g., WO 91/12882; WO 89/03429; and WO 92/03545.

Molecular conjugate vectors, such as the adenovirus chimeric vectorsdescribed in Michael, et al., (1993) J. Biol. Chem. 268:6866-6869 andWagner, et al., (1992) Proc. Natl. Acad. Sci. USA 89:6099-6103, can alsobe used for nucleic acid delivery or administration.

Members of the Alphavirus genus, such as, but not limited to, vectorsderived from the Sindbis, Semliki Forest, and Venezuelan EquineEncephalitis viruses, will also find use as viral vectors for deliveryand administration of nucleic acid molecules. For a description ofSindbis-virus derived vectors see, e.g., Dubensky, et al., (1996) J.Virol. 70:508-519; WO 95/07995; WO 96/17072; U.S. Pat. No. 5,843,723;and U.S. Pat. No. 5,789,245. See also WO 02/099035; and U.S. PublicationNo. 2003/018262.

Viral replicon particles can be used for delivery and administration ofnucleic acid and polypeptide molecules. For example, alphavirus repliconparticles, including chimeric alphavirus replicon particles, can be usedfor delivery and administration of nucleic acid and polypeptidemolecules. For a description of alphavirus replicon particle systemssee, e.g., WO 02/099035; U.S. Publication No. 2003/018262; WO 96/37616;U.S. Publication No. 2003/0119182; WO 03/023026; and WO 05/016961.

Nucleic acid and polypeptide molecules can also be delivered without aviral vector. For example, nucleic acid and/or polypeptide molecules canbe packaged in liposomes prior to delivery to the subject or to cellsderived therefrom. Lipid encapsulation is generally accomplished usingliposomes which are able to stably bind or entrap and retain nucleicacid.

The ratio of condensed DNA to lipid preparation can vary but willgenerally be around 1:1 (mg DNA:micromoles lipid), or more of lipid. Fora review of the use of liposomes as carriers for delivery of nucleicacids, see, Hug and Sleight, (1991) Biochim. Biophys. Acta. 1097:1-17;Straubinger, et al., (1983), Meth. Enzymol. 101:512-527.

Liposomal preparations for use in the present invention include cationic(positively charged), anionic (negatively charged) and neutralpreparations, with cationic liposomes particularly preferred. Cationicliposomes have been shown to mediate intracellular delivery of plasmidDNA (Felgner, et al., (1987) Proc. Natl. Acad. Sci. USA 84:7413-7416);mRNA (Malone, et al., (1989) Proc. Natl. Acad. Sci. USA 86:6077-6081);and purified transcription factors (Debs, et al., (1990) J. Biol. Chem.265:10189-10192), in functional form.

Cationic liposomes are readily available. For example,N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes areavailable under the trademark Lipofectin, from GIBCO BRL, Grand Island,N.Y. (See, also, Felgner, et al., (1987) Proc. Natl. Acad. Sci. USA84:7413-7416). Other commercially available lipids include (DDAB/DOPE)and DOTAP/DOPE (Boerhinger). Other cationic liposomes can be preparedfrom readily available materials using techniques well known in the art.See, e.g., Szoka, et al., (1978) Proc. Natl. Acad. Sci. USA75:4194-4198; WO 90/11092 for a description of the synthesis of DOTAP(1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes.

Similarly, anionic and neutral liposomes are readily available, such as,from Avanti Polar Lipids (Birmingham, Ala.), or can be easily preparedusing readily available materials. Such materials include phosphatidylcholine, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidyl glycerol (DOPG),dioleoylphoshatidyl ethanolamine (DOPE), among others. These materialscan also be mixed with the DOTMA and DOTAP starting materials inappropriate ratios. Methods for making liposomes using these materialsare well known in the art.

The liposomes can comprise multilammelar vesicles (MLVs), smallunilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs). Thevarious liposome-nucleic acid complexes are prepared using methods knownin the art. See, e.g., Straubinger, et al., (1983) Meth. Immunol.101:512-527; Szoka, et al., (1978) Proc. Natl. Acad. Sci. USA75:4194-4198; Papahadjopoulos, et al., (1975) Biochim. Biophys. Acta394:483-491; Wilson, et al., (1979) Cell 17:77-84); Deamer and Bangham,(1976) Biochim. Biophys. Acta 443:629-634; Ostro, et al., (1977)Biochem. Biophys. Res. Commun. 76:836-842; Fraley, et al., (1979) Proc.Natl. Acad. Sci. USA 76:3348-3352); Enoch and Strittmatter, (1979) Proc.Natl. Acad. Sci. USA 76:145-149; Fraley, et al., (1980) J. Biol. Chem.255:10431-10435; Szoka and Papahadjopoulos, (1978) Proc. Natl. Acad.Sci. USA 75:4194-4198; and Schaefer-Ridder, et al., (1982) Science215:166-168.

The nucleic acid and/or polypeptide molecules can also be delivered incochleate lipid compositions similar to those described byPapahadjopoulos, et al., (1975) Biochem. Biophys. Acta. 394:483-491.See, also, U.S. Pat. Nos. 4,663,161 and 4,871,488.

The nucleic acid and/or polypeptide molecules may also be encapsulated,adsorbed to, or associated with, particulate carriers. Examples ofparticulate carriers include those derived from polymethyl methacrylatepolymers, as well as microparticles derived from poly(lactides) andpoly(lactide-co-glycolides), known as PLG. See, e.g., Jeffery, et al.,(1993) Pharm. Res. 10:362-368; McGee, J P, et al., (1997)JMicroencapsul. 14(2):197-210; O'Hagan D T, et al., (1993) Vaccine11(2):149-154. Suitable microparticles may also be manufactured in thepresence of charged detergents, such as anionic or cationic detergents,to yield microparticles with a surface having a net negative or a netpositive charge. For example, microparticles manufactured with anionicdetergents, such as hexadecyltrimethylammonium bromide (CTAB), i.e.CTAB-PLG microparticles, adsorb negatively charged macromolecules, suchas DNA (see, e.g., WO 00/06123).

The vaccines will generally include one or more “pharmaceuticallyacceptable excipients or vehicles” such as water, saline, glycerol,ethanol, etc. Exemplary excipients also include, without limitation,those selected from the group consisting of carbohydrates, inorganicsalts, antimicrobial agents, antioxidants, surfactants, buffers, acids,bases, and combinations thereof. Additionally, auxiliary substances,such as wetting or emulsifying agents, pH buffering substances, and thelike, may be present in such vehicles. These foregoing pharmaceuticalexcipients along with other excipients are described in “Remington: TheScience & Practice of Pharmacy”, 19^(th) ed., Williams & Williams,(1995), the “Physician's Desk Reference”, 52^(nd) ed., MedicalEconomics, Montvale, N.J. (1998), and Kibbe, A. H., Handbook ofPharmaceutical Excipients, 3^(rd) Edition, American PharmaceuticalAssociation, Washington, D.C., 2000.

A carrier is optionally present which is a molecule that does not itselfinduce the production of antibodies harmful to the individual receivingthe composition. Suitable carriers are typically large, slowlymetabolized macromolecules such as proteins, polysaccharides, polylacticacids, polyglycolic acids, polymeric amino acids, amino acid copolymers,lipid aggregates (such as oil droplets or liposomes), and inactive virusparticles. Such carriers are well known to those of ordinary skill inthe art. Furthermore, the Env polypeptide may be conjugated to abacterial toxoid, such as toxoid from diphtheria, tetanus, cholera, etc.

Adjuvants may also be used to enhance the effectiveness of the vaccines.Such adjuvants include, but are not limited to: (1) aluminum salts(alum), such as aluminum hydroxide, aluminum phosphate, aluminumsulfate, etc.; (2) oil-in-water emulsion formulations (with or withoutother specific immunostimulating agents such as muramyl peptides (seebelow) or bacterial cell wall components), such as for example (a) MF59(International Publication No. WO 90/14837), containing 5% Squalene,0.5% Tween 80, and 0.5% Span 85 (optionally containing various amountsof MTP-PE (see below), although not required) formulated into submicronparticles using a microfluidizer such as Model 110Y microfluidizer(Microfluidics, Newton, Mass.), (b) SAF, containing 10% Squalane, 0.4%Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP (see below)either microfluidized into a submicron emulsion or vortexed to generatea larger particle size emulsion, and (c) Ribi™ adjuvant system (RAS),(Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene, 0.2% Tween80, and one or more bacterial cell wall components from the groupconsisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM),and cell wall skeleton (CWS), preferably MPL+CWS (Detox™); (3) saponinadjuvants, such as Stimulon™ (Cambridge Bioscience, Worcester, Mass.)may be used or particle generated therefrom such as ISCOMs(immunostimulating complexes); (4) Complete Freunds Adjuvant (CFA) andIncomplete Freunds Adjuvant (IFA); (5) cytokines, such as interleukins(IL-1, IL-2, etc.), macrophage colony stimulating factor (M-CSF), tumornecrosis factor (TNF), etc.; (6) detoxified mutants of a bacterialADP-ribosylating toxin such as a cholera toxin (CT), a pertussis toxin(PT), or an E. coli heat-labile toxin (LT), particularly LT-K63 (wherelysine is substituted for the wild-type amino acid at position 63)LT-R72 (where arginine is substituted for the wild-type amino acid atposition 72), CT-S109 (where serine is substituted for the wild-typeamino acid at position 109), and PT-K9/G129 (where lysine is substitutedfor the wild-type amino acid at position 9 and glycine substituted atposition 129) (see, e.g., International Publication Nos. WO93/13202 andWO92/19265); (7) MF59-CpG, (8) PLG-CpG, and (9) other substances thatact as immunostimulating agents to enhance the effectiveness of thecomposition.

Muramyl peptides include, but are not limited to,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acteyl-normuramyl-L-alanyl-D-isogluatme (nor-MDP),N-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-huydroxyphosphoryloxy)-ethylamine(MTP-PE), etc.

Typically, the vaccine compositions are prepared as injectables, eitheras liquid solutions or suspensions; solid forms suitable for solutionin, or suspension in, liquid vehicles prior to injection may also beprepared. The preparation also may be emulsified or encapsulated inliposomes for enhanced adjuvant effect, as discussed above. Thepharmaceutical preparation can also take other forms such as syrups,creams, ointments, tablets, powders, and the like. Other modes ofadministration are also included, such as pulmonary, rectal,transdermal, transmucosal, oral, intrathecal, subcutaneous,intra-arterial, and so forth.

As previously described, the compositions described herein (complexes,mimetics, antibodies) can be administered injected parenterally byintravenous injection, or less preferably by intramuscular or bysubcutaneous injection. Suitable formulation types for parenteraladministration include ready-for-injection solutions, dry powders forcombination with a solvent prior to use, suspensions ready forinjection, dry insoluble compositions for combination with a vehicleprior to use, and emulsions and liquid concentrates for dilution priorto administration, among others.

The vaccines will comprise a therapeutically effective amount of theEnv-CD4 mimetic complexes, or further complexes of these complexes, orantibodies directed to these complexes and any other of theabove-mentioned components, as needed. By “therapeutically effectiveamount” is meant an amount of a CD4 mimetic-complexed Env (e.g., gp120)protein that will induce a protective immunological response in theuninfected, infected or unexposed individual to whom it is administered.Such a response will generally result in the development in the subjectof a secretory, cellular and/or antibody-mediated immune response to thevaccine. Usually, such a response includes but is not limited to one ormore of the following effects; the production of antibodies from any ofthe immunological classes, such as immunoglobulins A, D, E, G or M; theproliferation of B and T lymphocytes; the provision of activation,growth and differentiation signals to immunological cells; expansion ofhelper T cell, suppressor T cell, and/or cytotoxic T cell.

Preferably, the effective amount is sufficient to bring about treatmentor prevention of disease symptoms. The exact amount necessary will varydepending on the subject being treated; the age and general condition ofthe individual to be treated; the capacity of the individual's immunesystem to synthesize antibodies; the degree of protection desired; theseverity of the condition being treated; the particular Env-CD4 complexselected and its mode of administration, among other factors. Anappropriate effective amount can be readily determined by one of skillin the art. A “therapeutically effective amount” will fall in arelatively broad range that can be determined through routine trials.

CD4 mimetics, complexes and antibodies can be injected or otherwiseadministered either subcutaneously, epidermally, intradermally,intramucosally such as nasally, rectally and vaginally,intraperitoneally, intravenously, orally or intramuscularly. Other modesof administration include oral and pulmonary administration,suppositories, needle-less injection, transcutaneous and transdermalapplications. Dosage treatment may be a single dose schedule or amultiple dose schedule. Administration may also be combined withadministration of peptides or other substances.

While the invention has been described in conjunction with the preferredspecific embodiments thereof, it is to be understood that the foregoingdescription as well as the examples which follow are intended toillustrate and not limit the scope of the invention. Other aspects,advantages and modifications within the scope of the invention will beapparent to those skilled in the art to which the invention pertains.

All articles, books, patents and other publications referenced hereinare hereby incorporated by reference in their entireties.

EXAMPLES

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

Example 1 Rational Design of CD4 Mimetics

Small molecules that act as CD4 mimetics as described herein wereobtained as follows. In a first step, based on known information aboutCD4 and CD4 mimetic, CD4M33 (Martin et al. (2003) Nat. Biotechnol.21(1):71-76), an in-house library of small molecules was examined forcompounds exhibiting structural similarity to CD4M33. Approximately 100compounds were identified in this first preliminary screening step.

In a second step, each of the 100 structurally identified compounds wasscreened for its ability to compete with CD4 binding to Env (gp120) aswell as its ability to induce the desired conformational change in theHIV Env protein to expose inducible epitopes. Both binding competitionand exposure of cryptic epitopes were analyzed using Biacore's SRPsystems. (Biacore Inc., Piscataway, N.J.), following the manufacturer'sinstructions.

As illustrated graphically in FIG. 1, compound 5 (ethyl1-[(4,6-dimethyl-1H-indol-2-yl)carbonyl]piperidine-4-carboxylate)inhibited binding of Env to the CD4 chip in comparison to when the smallmolecule was not present, indicating that the small molecule 5 competedwith the CD4 on the chip to bind to the Env protein.

Similarly, FIG. 2 illustrates that a complex of Env and small molecule 5exhibited enhanced binding to monoclonal antibody 17b in comparison toknown Env-sCD4 complexes (see Example 4 which follows). Illustrative CD4mimetic, 5, both competes with CD4 for binding to cryptic epitopes onEnv and upregulates binding to mAB 17b.

Thus, small molecule CD4 mimetics such as those described herein inducea conformational transformation of oligomeric (o-gp140) envelopes tothereby unmask cryptic epitopes close to co-receptor sites in the gp120subunit and efficiently increase co-receptor binding affinity indifferent gp120 envelopes.

Example 2 Unmasking Cryptic Epitopes of GP41 Subunit in OligomericEnvelopes

The CD4 mimetics identified as described above are also tested for theirability to induce a conformational transformation to expose crypticepitopes in oligomeric Env structures using SPR technology (Example 1)with 2F5 mAb or DP178 peptides (or congeners).

In instances in which Biacore screening indicates that gp41 epitopes areexposed upon binding of particular CD4 mimetics as described herein tooligomeric Env proteins, multiple mimetics may then be coupled toproduce novel bi-functional ligands, presenting increased potency inunmasking Env (gp41) epitopes. Novel chimeric oligomeric envelopes,complexed to such bi-functional ligand mimetics are also produced andtested. Candidate envelope proteins with superior exposure of gp120 andgp41 cryptic epitopes are subsequently tested in animals for theinduction of neutralizing antibodies.

Example 3 Production of Monoclonal Antibodies Targeting CrypticConserved Epitopes of Env

Selected Env-CD4 mimetic complex immunogens are injected in rats or miceto prepare monoclonal antibodies according to standard procedures.Clones are screened in ELISA against CD4 miniprotein-gp120 complex, CD4miniprotein-o-gp 140, gp120 and o-gp140 alone and CD4M33 mimetic aswell. Clones exhibiting highest affinity for complexes as compared toenvelopes alone are further tested in Biacore, as described inExample 1. Clones scoring positive in Biacore against the CD4M33-gp120and or CD4M33-o-gp140 complexes are selected and used for bulkproduction of ascites fluids.

Example 4 Production of Monoclonal Antibodies Targeting CrypticConserved Epitopes of Env

Surface plasmon resonance assays were performed using a BIACORE 3000optical biosensor system (Biacore AB, Uppsala, Sweden) with simultaneousmonitoring of relevant flow cells. To perform the kinetic study of thebinding of HIV env to sCD4, sCD4 was immobilized using amine couplingonto CM5 sensor chip to attain 4000 response units. To perform thekinetic study of the binding of HIV env to the monoclonal antibody 17b,antibodies were immobilized using amine coupling onto a CM5 sensor chipto attain 2000 response units. Using PBS buffer (pH 7.4) with 0.05%Tween 20 and 3% DMSO, association was assessed by passing gp120SF162(GenBank P03378; Sanchez-Pescador et al. (1985) Science, 227(4686):484-492), with and without various controls or small molecules, over thechip surface at a flow rate of 25 μl/min. The concentration ofgp120SF162 used was 1 uM for all samples. As a negative control, acontrol surface coupled to 2000 response units of BSA (Pierce) was used.A buffer only control was subtracted from all sensograms. Responses weremeasured by assessing the maximum RUs (response units) obtained at thepeak of the binding curve.

The percentage upregulation of 17b was determined by dividing the peakRU observed for the Env polypeptide/compound complex divided by the peakRU for the Env polypeptide alone.

The following compounds in the table below were found to exhibit atleast a 1% upregulation of 17b.

TABLE 1 Compound Structure Name 1

1-[2-(5-methoxy-1H-indol-3- yl)-2-oxoethyl]piperidine-4- carboxamide 2

2-(4-benzylpiperazin-1-yl)-1- (5-methoxy-1H-indol-3- yl)ethanone 3

{1-[(4,6-dimethyl-1H-indol- 2-yl)carbonyl]piperidin-4- yl}(4-fluorophenyl)methanone 4

1-[2-(5-methoxy-1,2- dimethyl-1H-indol-3-yl)-1- methyl-2-oxoethyl]piperidine-4- carboxamide 5

ethyl 1-[(4,6-dimethyl-1H- indol-2- yl)carbonyl]piperidine-4-carboxylate 6

6-{[4-(2,6- difluorobenzoyl)piperazin-1- yl]carbonyl}-1H-indole 7

2-{[4-(1H-indol-6- ylcarbonyl)piperazin-1- yl]carbonyl}phenyl acetate 8

6-{[4-(3,4,5- trimethoxybenzoyl)piperazin- 1-yl]carbonyl}-1H-indole 9

1-[2-(5-methoxy-1,2- dimethyl-1H-indol-3-yl)-2- oxoethyl]piperidine-4-carboxamide 10

4-{[4-(2-furoyl)piperazin-1- yl]carbonyl}-9H-fluoren-9- one

Example 5 Neutralizing Antibody Production Using Small Molecule CD4Mimetics or CD4 Mimetic-Env Complexes

Rabbits

Groups of 4-5 or more rabbits each are immunized with small molecule CD4mimetics and/or complexes of Env-CD4 mimetics at 0, 4, 12 and 24 weeks.Sera are collected biweekly and analyzed against, e.g., SF2 or SF162gp120 in an ELISA. The results from these rabbit studies will revealwhich animals mount a strong immune response against gp120.

Monkeys

Groups of 5 or more monkeys are immunized with CD4 mimetics and/orEnv-CD4 mimetic complexes with adjuvant along with control groups of Envprotein. Complexes and are made with monomeric and oligomeric forms ofSF162 Env with and without V2-deletes and the antibody responsescompared. Immunization schedules are at 0, 4, and 24 week immunizations.When warranted, an additional booster may be included at later timepoints

Example 6 Additional Screening of Small Molecule CD4 Mimetics for CD4Binding Inhibition And/or Upregulation of CD4 Inducible Epitopes

Additional candidate small molecules were screened for CD4 bindinginhibition and upregulation of CD4 inducible epitopes, e.g., mABs 17band 48D, by HPLC and BIAcore methods as described in Examples 1 and 4above. Each of the compounds in Table 5 below was screened three timesby both methods using different lots of both gp120 and antibodies. Therounds of screening tests were additionally carried out on differentdates.

CD4 binding competition was examined in the free solution state by HPLC.Various candidate small molecules were each mixed with gp120SF162 andthe resulting solution was incubated for 1 hour at 37° C.Fluorescently-labelled CD4, CD4 488, was then added to each incubatedsolution, and the resulting mixture was analyzed by size exclusion HPLC(SEC-HPLC), using fluorescent signal detection. The extent ofcompetition with CD4 was determined as follows. For small moleculecandidate samples in which the fluorescent signal was lower than thefluorescent signal of the control (gp120 alone), the small molecule wasdetermined to exhibit competitive binding with CD4. For small moleculecandidate samples in which the fluorescent signal was essentiallyunchanged or normal relative to the control, the small molecule wasdetermined to exhibit no competitive binding with CD4, i.e., the smallmolecule candidate was not classified as an inhibitor of CD4-binding toEnv.

Based upon these additional screening studies, numerous small moleculeCD4 mimetics were identified. In sum, three different classes of smallmolecule CD4 mimetics were identified: (i) molecules that inhibit CD4binding, (ii) molecules that upregulate CD4 inducible epitopes (17b and48D), and (iii) molecules that both inhibit CD4 binding and upregulateCD4 inducible epitopes.

Tables 2-4 below demonstrate the chemical structures and molecularweights of representative small molecule CD4 mimetics, classifiedaccording to their CD4-inhibitory binding capability as well as theirability to upregulate certain CD4 inducible epitopes.

Supporting screening data for illustrative small molecule CD4 mimeticsin accordance with the invention is presented as FIG. 3.

TABLE 2 Compounds that inhibit (compete with) CD4 but do not upregulate17b. Com- pound Structure Name Mol Formula MW 11

1-[4-(1H-indole-6-carbonyl)- piperazin-1-yl]-2,2-diphenyl- ethanone C₂₇H₂₅ N₃ O₂ 423.5 8

[4-(1H-Indole-6-carbonyl)- piperazin-1-yl]-(3,4,5-trimethoxy-phenyl)-methanone C₂₃ H₂₅ N₃ O₅ 423.5 12

[4-(1H-Indol-3-yl)-piperidin-1- yl]-(3,4,5-trimethoxy-phenyl)- methanoneC₂₃ H₂₆ N₂ O₄ 394.5 13

(5-Benzyloxy-3-methyl-1H- indol-2-yl)-(4-methyl-piperazin-1-yl)-methanone C₂₂ H₂₅ N₃ O₂ 363.5 14

(5-Benzyloxy-3-methyl-1H- indol-2-yl)-morpholin-4-yl- methanone C₂₁ H₂₂N₂ O₃ 350.4 15

2-{2-[2-(2-Methyl-1H-indol-3- yl)-2-oxo-ethyl]-1,2,3,4-tetrahydro-isoquinolin-1- ylmethyl}-isoindole-1,3-dione C₂₉ H₂₅ N₃ O₃463.5 16

1-[2-(2-Methyl-propenyl)-1H- indol-3-yl]-2-morpholin-4-yl-ethane-1,2-dione C₁₈ H₂₀ N₂ O₃ 312.4 17

(5-Methoxy-1,2-dimethyl-1H- indol-3-yl)-(4-methyl-piperazin-1-yl)-methanone C₁₇ H₂₃ N₃ O₂ 301.4 18

2-[1-(4-Chloro-benzoyl)-5- methoxy-2-methyl-1H-indol-3-yl]-1-(4-phenyl-piperazin-1-yl)- ethanone C₂₉ H₂₈ Cl N₃ O₃ 502.0

TABLE 3 Compounds that upregulate 17b but do not inhibit (compete with)CD4. Com- pound Structure Name Mol Formula MW 2

2-(4-Benzyl-piperazin-1-yl)-1- (5-methoxy-1H-indol-3-yl)- ethanone C₂₂H₂₅ N₃ O₂ 363.5 20

2-{2-[2-(1H-Indol-3-yl)-2-oxo- ethyl]-1,2,3,4-tetrahydro-isoqunolin-1-ylmethyl}- isoindole-1,3-dione C₂₈ H₂₃ N₃ O₃ 449.5 21

1-[2-(5-Methoxy-1,2-dimethyl- 1H-indol-3-yl)-2-oxo-ethyl]-piperidine-4-carboxylic acid amide C₁₉ H₂₅ N₃ O₃ 343.4 22

4-[2-Hydroxy-2-(5-methoxy-1H- indol-3-yl)-ethyl]-piperazine-1-carboxylic acid ethyl ester C₁₈ H₂₅ N₃ O₄ 347.4 23

1-[2-(5-Methoxy-1H-indol-3-yl)- 2-oxo-ethyl]-piperidine-4- carboxylicacid ethyl ester C₁₉ H₂₄ N₂ O₄ 344.4

TABLE 4 Compounds that inhibit (compete with) CD4 and upregulate 17b.Compound Structure Name Mol Formula MW 19

1-(1H-lndol-3-yl)-2-piperidin-1- yl-ethane-1,2-dione C₁₅ H₁₆ N₂ O₂ 256.320

[4-(1H-Indol-3-ylmethyl)- piperazin-1-yl]-{3,4,5-trimethoxy-phenyl)-methanone C₂₃ H₂₇ N₃ O₄ 409.5 21

1-(1H-Indol-3-yl)-2-morpholin- 4-yl-ethane-1,2-dione C₁₄ H₁₄ N₂ O₃ 258.322

(5-Benzyloxy-3-methyl-1H- indol-2-yl)-piperidin-1-yl- methanone C₂₂ H₂₄N₂ O₂ 348.4 23

Furan-2-carboxylic acid (1- benzyl-piperidin-4-yl)-(2,3-dimethyl-1H-indol-7-yl)-amide C₂₇ H₂₉ N₃ O₂ 427.5 24

(5-Benzyloxy-3-methyl-1H- indol-2-yl)-(4-phenyl-piperazin1-yl)-methanone C₂₇ H₂₇ N₃ O₂ 425.5 25

[4-(1H-Indol-3-ylmethyl)- piperazin-1-yl]-(2-methoxy- phenyl)-methanoneC₂₁ H₂₃ N₃ O₂ 349.4 26

2-[4-(2-Methoxy-phenyl)- piperazin-1-yl]-1-(2-methyl-1H-indol-3-yl)-ethanone C₂₂ H₂₅ N₃ O₂ 363.5 27

2-{2-[2-Hydroxy-2-(5-methoxy- 1H-indol-3-yl)-ethyl]-1,2,3,4-tetrahydro-isoquinolin-1- ylmethyl}-isoindole-1,3-dione C₂₉ H₂₇ N₃ O₄481.5 28

2-[4-(2-Hydroxy-ethyl)- piperazin-1-yl]-1-(5-methoxy-1H-indol-3-yl)-ethanone C₁₇ H₂₃ N₃ O₃ 317.4 29

2-{2-[2-(5-Methoxy-1H-indol-3- yl)-2-oxo-ethyl]-1,2,3,4-tetrahydro-isoquinolin-1- ylmethyl}-isoindole-1,3-dione C₂₉ H₂₅ N₃ O₄479.5 30

{ 4-[2-(5-Methoxy-1H-indol-3- yl)-2-oxo-ethyl]-piperazin-1-yl}- aceticacid methyl ester C₁₈ H₂₃ N₃ O₄ 345.4 1

1-[2-(5-methoxy-1H-indol-3-yl)-2-oxoethyl]piperidine-4- carboxamide 2

2-(4-benzylpiperazin-1-yl)-1-(5-methoxy-1H-indol-3- yl)ethanone 3

{1-[(4,6-dimethyl-1H-indol-2-yl)carbonyl]piperidin-4-yl}(4-fluorophenyl)methanone 4

1-[2-(5-methoxy-1,2-dimethyl-1H-indol-3-yl)-1-methyl-2-oxoethyl]piperidine-4-carboxamide 5

ethyl 1-[(4,6-dimethyl-1H-indol-2-yl)carbonyl]piperidine- 4-carboxylate6

6-{[4-(2,6-difluorobenzoyl)piperazin-1-yl]carbonyl}-1H- indole 7

2-{[4-(1H-indol-6-ylcarbonyl)piperazin-1- yl]carbonyl}phenyl acetate 8

6-{[4-(3,4,5-trimethoxybenzoyl)piperazin-1-yl]carbonyl}- 1H-indole 9

1-[2-(5-methoxy-1,2-dimethyl-1H-indol-3-yl)-2-oxoethyl]piperidine-4-carboxamide 10

4-{[4-(2-furoyl)piperazin-1-yl]carbonyl}-9H-fluoren-9-one

Example 7 Mucosal Challenge Following Administration of Env-CD4-InvasinComplexes

Evaluation of Env-CD4 mimetic complexes for their ability to conferprotection against heterologous challenge in rhesus macaques is tested.Specifically, combinations of these complexes with various adjuvants areadministered to macaques and responses (e.g., virus load, antibodyand/or T cell responses) of the animals evaluated pre- andpost-challenge with mucosally SHIV challenge. The results from both thepre-challenge (immunogenicity) and post-challenge arms should providedata that will reveal which candidate immunogens and deliveries warrantfurther evaluation in macaques. The potency and breadth of the observedimmune responses are considered as well as protective efficacy.

A. Cellular Immune Responses

The cellular immune parameters monitored regularly include enumerationof antigen-specific T-helper responses (IFN-γ, IL-2 and IL-4) asmeasured by ELISpot and lymphocyte proliferation (LP) responses. Thekinetics of T-cell responses will primarily be measured by enumeratingthe number of CD4+ and CD8+ T cells that secrete IFN-γ in response tospecific peptides. If particular (matching) MHC specific tetramerreagents are not available, then the majority of assays will byperformed by CD8+ ELISpot assays with overlapping pools of peptides,from which epitope specificities are determined. Classical bulk lysis(Chromium release) assays are performed as confirmatory assays.Additional supplementary assays such as flow cytometric assays tomeasure antigen-specific intracellular cytokines in T-cell subsets, andtetramer analysis to measure antigen-specific CD8+ T-cell populations inanimals with MHC specificities that match available tetramers will alsobe performed on a subset of animals.

Assays for the measurement of cellular immune responses in the mucosalwill include ELISpot, measurements of antibody-secreting cells (ASC),and FACS analysis of antigen-specific intracellular cytokines in mucosalT-cells.

B. Humoral/Neutralizing Antibody Responses

Antibody responses and neutralization assays are performed as describedabove and/or samples are sent to outside vendor for evaluation (e.g.,Virologic, Inc).

C. Measurements of Vaccine Efficacy Post-Challenge

The Env-CD4 mimetics complexes that give strong and broadneutralization, complemented by strong CD4 and CD8 T cell responses, aretested by homologous and heterologous systemic (IV) and mucosal (IR)challenges. Over the last ten years this has proven to be the mostreliable challenge dose where protection can be achieved with allcontrols contacting disease. Challenge is performed 8 weeks after thefinal immunization, except during the final year of study whenchallenges are performed in parallel groups of monkeys when half of thedesignated groups are challenged at 8 months after the finalimmunization.

The post-challenge measurements of vaccine efficacy to be evaluated aresummarized in Table 5. The plasma and PBMC are collected for analysis(e.g., virological and FACS) at two-week intervals for the first twomonths after challenge and at monthly intervals thereafter.

Plasma virus load analysis is performed as well as a detailed analysisof any persistent low copy proviral infection of PBMC or lymph nodecells. Detailed FACS analysis provide evidence of T-cell activationand/or CD4+ T cell loss and disease progression. Immunoblot assays andELISA for non-vaccine viral antigens are used to determine whetherseroconversion has occurred.

Protected animals are identified after taking all these parameters intoaccount. “Protection against infection” is defined as no indication ofvirus infection by any of the virologic (and serologic) sensitive assayslisted in Table 5. “Partial protection” is defined by significantreductions in the virus load as measured by quantitative virusisolation, levels of viral RNA, frequency of detection of proviral DNAin PBMC and lymph nodes (LN). “Protection from disease” is definedprimarily by reductions in the decline of CD4+ T cells, fewer or lesssevere signs and symptoms, survival, and secondarily, by reductions invirus load, and the robustness of the post-challenge immune responses.

TABLE 5 Post-Challenge Measurements of Vaccine Efficacy Measurements ofVirus Load Immune Status/Disease Progression Plasma RNA (quantitativePCR) FACS (CD3, CD4, CD8, CD16, HLA-DR, CD20) Proviral DNA (nestedPCR/PBMC Immunoblot and ELISA and LN) (seroconversion) Quantitativevirus isolation Clinical signs and symptoms (PBMC) Ag specific IFN-γ,IL-2, IL-4, ELISPot ICS for IL-2 loss in CD4 subsets

The relative merit of a vaccine regimen for future study are decidedbased on the outcome of the challenges and the observed potency,breadth, and durability of the immune responses pre- and post-challenge.In addition to the magnitude of the antigen-specific IL-2 responses(ELISPot) before challenge, the loss of Ag-specific IL-2 producing CD4T-cell subsets as measured by ICS is prognostic of failure tocontain/control virus load after challenge (see, also, Ogg et al. (1998)Science 279(5359):2103-2106; Oldstone et al. (1997) Virol.234(2):179-185).

1. A method of eliciting an immune response in a subject, the methodcomprising administering to the an Env polypeptide and a CD4 mimetic,wherein the Env protein and CD4 mimetic are administered simultaneously,sequentially in any order, separately, or as a complex, and wherein theCD4 mimetic is selected from the group consisting of{1-[(4,6-dimethyl-1H-indol-2-yl)carbonyl]piperidin-4-yl}(4-fluorophenyl)methanoneand ethyl1-[(4,6-dimethyl-1H-indol-2-yl)carbonyl]piperidine-4-carboxylate.
 2. Themethod of claim 1, wherein the Env polypeptide and the CD4 mimetic areadministered intravenously, intramuscularly or subcutaneously.
 3. Themethod of claim 1, wherein the immune response is a cellular productionof antibodies to CD41.
 4. The method of claim 1, wherein the Envpolypeptide is a gp120 polypeptide.
 6. The method of claim 1, whereinthe small molecule CD4 mimetic is{1-[(4,6-dimethyl-1H-indol-2-yl)carbonyl]piperidin-4-yl}(4-fluorophenyl)methanone.7. The method of claim 1, wherein the small molecule CD4 mimetic isethyl 1-[(4,6-dimethyl-1H-indol-2-yl)carbonyl]piperidine-4-carboxylate.8. A method of producing an antibody that binds to a cryptic epitope ofan Env polypeptide, the method comprising administering an Envpolypeptide and a CD4 mimetic, wherein the Env protein and CD4 mimetic,wherein the CD4 mimetic is selected from the group consisting of{1-[(4,6-dimethyl-1H-indol-2-yl)carbonyl]piperidin-4-yl}(4-fluorophenyl)methanoneand ethyl1-[(4,6-dimethyl-1H-indol-2-yl)carbonyl]piperidine-4-carboxylate underconditions effective to form antibodies in the subject.
 9. The method ofclaim 8, further comprising the step of isolating the antibodies formedby the subject.
 10. The method of claim 8, wherein the antibodies formedare neutralizing antibodies.
 11. The method of claim 8, wherein theantibodies formed are monoclonal antibodies.
 12. The method of claim 8,wherein the antibodies are polyclonal antibodies.
 13. The method ofclaim 8, wherein the Env polypeptide is a gp120 polypeptide.
 14. Themethod of claim 8, wherein the small molecule CD4 mimetic is{1-[(4,6-dimethyl-1H-indol-2-yl)carbonyl]piperidin-4-yl}(4-fluorophenyl)methanone.15. The method of claim 8, wherein the small molecule CD4 mimetic isethyl 1-[(4,6-dimethyl-1H-indol-2-yl)carbonyl]piperidine-4-carboxylate.